Search

High amounts of glycosaminoglycans (GAG) such as hyaluronan (HA) occur in connective tissues. There is nowadays increasing evidence that a "sulfation code" exists which mediates numerous GAG functions. High molecular weight and inhomogeneity of GAG, however, aggravated detailed studies. Thus, synthetic oligosaccharides were urgently required. We will review here chemoenzymatic and analytic strategies to provide defined sulfated and anomerically modified GAG oligosaccharides of the HA type. Representative studies of protein/GAG interactions by (bio)chemical and biophysical methods are reported yielding novel insights into GAG-protein binding. Finally, the biological conclusions and in vivo applications of defined sulfated GAG oligosaccharides will be discussed., (© 2021 Jürgen Schiller et al., published by De Gruyter, Berlin/Boston.)

Angiogenesis is an important physiological process playing a crucial role in wound healing and cancer progression. Vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) are key players in angiogenesis. Based on previous findings regarding the modulation of VEGF activity by glycosaminoglycans (GAG), here we explore the interaction of hyaluronan (HA)-based GAG with PDGF and its receptor PDGFR-β by applying molecular modeling and dynamics simulations in combination with surface plasmon resonance (SPR). Computational analysis on the interaction of oligo-hyaluronan derivatives with different sulfation pattern and functionalization shows that these GAG interact with PDGF in relevant regions for receptor recognition, and that high sulfation as well as modification with the TAMRA group convey stronger binding. On the other hand, the studied oligo-hyaluronan derivatives are predicted to scarcely recognize PDGFR-β. SPR results are in line with the computational predictions regarding the binding pattern of HA tetrasaccharide (HA4) derivatives to PDGF and PDGFR-β. Furthermore, our experimental results also show that the complexation of PDGF to PDGFR-β can be modulated by HA4 derivatives. The results found open the path for considering HA4 derivatives as potential candidates to be exploited for modulation of the PDGF/PDGFR-β signaling system in angiogenesis and related disease conditions., (© 2021 Kanagasabai Balamurugan et al., published by De Gruyter, Berlin/Boston.)

The delivery of chemotactic signaling molecules via customized biomaterials can effectively guide the migration of cells to improve the regeneration of damaged or diseased tissues. Here, we present a novel biohybrid hydrogel system containing two different sulfated glycosaminoglycans (sGAG)/sGAG derivatives, namely either a mixture of short heparin polymers (Hep-Mal) or structurally defined nona-sulfated tetrahyaluronans (9s-HA4-SH), to precisely control the release of charged signaling molecules. The polymer networks are described in terms of their negative charge, i.e. the anionic sulfate groups on the saccharides, using two parameters, the integral density of negative charge and the local charge distribution (clustering) within the network. The modulation of both parameters was shown to govern the release characteristics of the chemotactic signaling molecule SDF-1 and allows for seamless transitions between burst and sustained release conditions as well as the precise control over the total amount of delivered protein. The obtained hydrogels with well-adjusted release profiles effectively promote MSC migration in vitro and emerge as promising candidates for new treatment modalities in the context of bone repair and wound healing., (© 2021 Sebastian Kühn et al., published by De Gruyter, Berlin/Boston.)

The generation of bioactive molecules from inactive precursors is a crucial step in the chemical evolution of life, however, mechanistic insights into this aspect of abiogenesis are scarce. Here, we investigate the protein-catalyzed formation of antivirals by the 3C-protease of enterovirus D68. The enzyme induces aldol condensations yielding inhibitors with antiviral activity in cells. Kinetic and thermodynamic analyses reveal that the bioactivity emerges from a dynamic reaction system including inhibitor formation, alkylation of the protein target by the inhibitors, and competitive addition of non-protein nucleophiles to the inhibitors. The most active antivirals are slowly reversible inhibitors with elongated target residence times. The study reveals first examples for the chemical evolution of bio-actives through protein-catalyzed, non-enzymatic C-C couplings. The discovered mechanism works under physiological conditions and might constitute a native process of drug development., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Dishevelled (Dvl) proteins are important regulators of the Wnt signalling pathway, interacting through their PDZ domains with the Wnt receptor Frizzled. Blocking the Dvl PDZ-Frizzled interaction represents a potential approach for cancer treatment, which stimulated the identification of small-molecule inhibitors, among them the anti-inflammatory drug Sulindac and Ky-02327. Aiming to develop tighter binding compounds without side effects, we investigated structure-activity relationships of sulfonamides. X-ray crystallography showed high complementarity of anthranilic acid derivatives in the GLGF loop cavity and space for ligand growth towards the PDZ surface. Our best binding compound inhibits Wnt signalling in a dose-dependent manner as demonstrated by TOP-GFP assays (IC 50 ∼ 50   µ M ) and Western blotting of β -catenin levels. Real-time PCR showed reduction in the expression of Wnt-specific genes. Our compound interacted with Dvl-1 PDZ (K D = 2.4   µ M ) stronger than Ky-02327 and may be developed into a lead compound interfering with the Wnt pathway., Competing Interests: The authors declare that they have no conflict of interest., (Copyright: © 2021 Nestor Kamdem et al.)

The rapid generation and modification of macrocyclic peptides in medicinal chemistry is an ever-growing area that can present various synthetic challenges. The reaction between N-terminal cysteine and 2-cyanoisonicotinamide is a new biocompatible click reaction that allows rapid access to macrocyclic peptides. Importantly, 2-cyanoisonicotinamide can be attached to different linkers directly during solid-phase peptide synthesis. The synthesis involves only commercially available precursors, allowing for a fully automated process. We demonstrate the approach for four cyclic peptide ligands of the Zika virus protease NS2B-NS3. Although all peptides display the substrate recognition motif, the activity strongly depends on the linker length, with the shortest cyclization linker corresponding to highest activity ( K i = 0.64 μM). The most active cyclic peptide displays affinity 78 times higher than that of its linear analogue. We solved a crystal structure of the proteolytically cleaved ligand and synthesized it by applying the presented chemistry to peptide ligation., Competing Interests: The authors declare no competing financial interest., (© 2021 American Chemical Society.)

Glycosaminoglycans (GAG) as long, unbranched polysaccharides are major components of the extracellular matrix. Many studies provided additional evidence of a specific binding between mediators and sulfated GAG, at which the sulfation code-which means the number and positions of sulfate groups along the polysaccharide chain-plays an important role. GAG from natural sources are very inhomogeneous regarding their sulfation patterns and molecular weight. Additionally, there is a high risk of contamination. This results in a growing interest in the careful characterization of native GAG and the synthesis of artificial GAG. Additionally, chemically oversulfated GAG analogues show many favorable properties. However, the structural characterization of these carbohydrates by mass spectrometry remains challenging. One significant problem is the sulfate loss during the ionization, which increases with the number of sulfate residues. We used the sulfated pentasaccharide fondaparinux as model substance to optimize sample preparation and measurement conditions, compared different established desalination methods and already existing protocols for sulfated oligosaccharides, and investigated their impact on the quality of the mass spectra. After optimization of the measurement conditions, we could establish a gentle and fast protocol for the mass spectrometry characterization of (fully) sulfated, artificial GAG-like oligosaccharides with minimized sulfate loss in the positive and negative ion mode. Here, the negative ion mode was more sensitive in comparison with the positive one, and fondaparinux species with sulfate loss were not detectable under the optimized conditions in the positive ion mode., (© 2020 The Authors. Journal of Mass Spectrometry published by John Wiley & Sons Ltd.)

Supporting the wound healing process by sending the appropriate cytokine signals can shorten healing time and overcome chronic inflammation syndromes. Even though adhesion peptides consisting of Arg-Gly-Asp (RGD) are commonly used to enhance cell-surface interactions, peptide-mediated cytokine delivery has not been widely exploited so far. Cytokines interact with high affinity with their cognitive receptors but also with sulfated glycosaminoglycans (GAGs), both of which form a base for incorporation of cytokines into functional biomaterials. Here, we report on a mussel-derived surface coating as a prospective cytokine delivery system using covalently bound heparin mimetics, receptor-derived chemokine-binding peptides, and heparin-binding peptides (HBP). The latter enabled non-covalent immobilization of heparin on the surface followed by chemokine binding and release, whereas the former allowed direct non-covalent chemokine immobilization. The peptide displayed excellent binding to custom-made polystyrene 96-well plates, enabling convenient testing of several compounds. Released chemokine successfully induced migration in Jurkat cells, especially for the non-covalent heparin immobilization approach using HBPs as evaluated in a transwell assay. In comparison, heparin-mimetic coatings, comprised of sulfated peptides and GAG derivatives, proved less efficient with respect to amount of immobilized chemokine and migratory response. Thus, our study provides a roadmap for further rational optimization and translation into clinics., (© 2020 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons Ltd.)

Toll-like receptors (TLRs) build the first barrier in the innate immune response and therefore represent promising targets for the modulation of inflammatory processes. Recently, the pyrogallol-containing TLR2 antagonists CU-CPT22 and MMG-11 were reported; however, their 1,2,3-triphenol motif renders them highly susceptible to oxidation and excludes them from use in extended experiments under aerobic conditions. Therefore, we have developed a set of novel TLR2 antagonists (1-9) based on the systematic variation of substructures, linker elements, and the hydrogen-bonding pattern of the pyrogallol precursors by using chemically robust building blocks. The novel series of chemically stable and synthetically accessible TLR2 antagonists (1-9) was pharmacologically characterized, and the potential binding modes of the active compounds were evaluated structurally. Our results provide new insights into structure-activity relationships and allow rationalization of structural binding characteristics. Moreover, they support the hypothesis that this class of TLR ligands bind solely to TLR2 and do not directly interact with TLR1 or TLR6 of the functional heterodimer. The most active compound from this series (6), is chemically stable, nontoxic, TLR2-selective, and shows a similar activity with regard to the pyrogallol starting points, thus indicating the variability of the hydrogen bonding pattern., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Toll-like receptor 2 (TLR2) and TLR8 are involved in the recognition of bacterial and viral components and are linked not only to protective antimicrobial immunity but also to inflammatory diseases. Recently, increasing attention has been paid to the receptor crosstalk between TLR2 and TLR8 to fine-tune innate immune responses. In this study, we report a novel dual TLR2/TLR8 antagonist, compound 24 that was developed by a modeling-guided synthesis approach. The modulator was optimized from the previously reported 1,3-benzothiazole derivative, compound 8. Compound 24 was pharmacologically characterized for the ability to inhibit TLR2- and TLR8-mediated responses in TLR-overexpressing reporter cells and THP-1 macrophages. The modulator showed high efficacy with IC 50 values in the low micromolar range for both TLRs, selectivity towards other TLRs and low cytotoxicity. At TLR2, a slight predominance for the TLR2/1 heterodimer was found in reporter cells selectively expressing TLR2/1 or TLR2/6 heterodimers. Concentration ratio analysis in the presence of Pam 3 CSK 4 or Pam 2 CSK 4 indicated non-competitive antagonist behavior at hTLR2. In computational docking studies, a plausible alternative binding mode of compound 24 was predicted for both TLR2 and TLR8. Our results provide evidence that it is feasible to simultaneously and selectively target endosomal- and surface-located TLRs. We identified a small-molecule dual TLR2/8 antagonist that may serve as a valuable pharmacological tool to decipher the role of TLR2/8 co-signaling in inflammation., 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 © 2020 Elsevier Inc. All rights reserved.)

The flexible variation of peptidomimetics is of great interest for the identification of optimized protein ligands. Here we present a general concept for introducing side-chain modifications into peptides using triarylphosphonium amino acids. Building blocks 4a and 4b are activated for amidation and incorporated into stable peptides. The obtained phosphoranylidene peptides undergo Wittig olefinations and 1,3-dipolar cycloaddition reactions, yielding peptidomimetics with vinyl ketones and 5-substituted 1,2,3-triazoles as non-native peptide side chains.

The pivotal role of viral proteases in virus replication has already been successfully exploited in several antiviral drug design campaigns. However, no efficient antivirals are currently available against flaviviral infections. In this study, we present lead-like small molecule inhibitors of the Zika Virus (ZIKV) NS2B-NS3 protease. Since only few nonpeptide competitive ligands are known, we take advantage of the high structural similarity with the West Nile Virus (WNV) NS2B-NS3 protease. A comparative modeling approach involving our in-house software PyRod was employed to systematically analyze the binding sites and develop molecular dynamics-based 3D pharmacophores for virtual screening. The identified compounds were biochemically characterized revealing low micromolar affinity for both ZIKV and WNV proteases. Their lead-like properties together with rationalized binding modes represent valuable starting points for future lead optimization. Since the NS2B-NS3 protease is highly conserved among flaviviruses, these compounds may also drive the development of pan-flaviviral antiviral drugs., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

Glycosaminoglycans (GAGs) are a physio- and pharmacologically highly relevant class of complex saccharides, possessing a linear sequence and strongly acidic character. Their repetitive linear core makes them seem structurally simple at first glance, yet differences in sulfation and epimerization lead to an enormous structural diversity with only a few GAGs having been successfully characterized to date. Recent infrared action spectroscopic experiments on sulfated mono- and disaccharide ions show great promise. Here, we assess the potential of two types of gas-phase action spectroscopy approaches in the range from 1000 to 1800 cm -1 for the structural analysis of complex GAG oligosaccharides. Synthetic tetra- and pentasaccharides were chosen as model compounds for this benchmark study. Utilizing infrared multiple photon dissociation action spectroscopy at room temperature, diagnostic bands are largely unresolved. In contrast, cryogenic infrared action spectroscopy of ions trapped in helium nanodroplets yields resolved infrared spectra with diagnostic features for monosaccharide composition and sulfation pattern. The analysis of GAGs could therefore significantly benefit from expanding the conventional MS-based toolkit with gas-phase cryogenic IR spectroscopy. Graphical abstract.

Pathological healing characterized by abnormal angiogenesis presents a serious burden to patients' quality of life requiring innovative treatment strategies. Glycosaminoglycans (GAG) are important regulators of angiogenic processes. This experimental and computational study revealed how sulfated GAG derivatives (sGAG) influence the interplay of vascular endothelial growth factor (VEGF) 165 and its heparin-binding domain (HBD) with the signaling receptor VEGFR-2 up to atomic detail. There was profound evidence for a HBD-GAG-HBD stacking configuration. Here, the sGAG act as a "molecular glue" leading to recognition modes in which sGAG interact with two VEGF 165 -HBDs. A 3D angiogenesis model demonstrated the dual regulatory role of high-sulfated derivatives on the biological activity of endothelial cells. While GAG alone promote sprouting, they downregulate VEGF 165 -mediated signaling and, thereby, elicit VEGF 165 -independent and -dependent effects. These findings provide novel insights into the modulatory potential of sGAG derivatives on angiogenic processes and point towards their prospective application in treating abnormal angiogenesis.

Controlled release of active biomolecules is an attractive approach to modulate chemotactic gradients and accordingly the recruitment of cells, e.g. endothelial progenitor cells to improve wound healing or stimulate angiogenesis after myocardial infarction. Here, we developed variants of hCXCL12, also named stromal cell-derived factor 1α, a chemokine that activates the CXCR4 and consequently recruits tissue specific stem and progenitor cells. hCXCL12 variants were designed to bind to glycosaminoglycans (GAGs) with different affinities in order to modulate its release. Sixteen analogs were recombinantly produced, characterized, and tested for their GAG-binding property. The most promising variants hCXCL12 K24/K27/R41/R47A and hCXCL12 Q48K were used for release studies from starPEG-heparin-hydrogels. The reduced GAG affinity led to a fast release of hCXCL12 K24/K27/R41/R47A, whereas hCXCL12 Q48K was slowly released over 2 weeks due to its increased binding strength compared to wild type hCXCL12. Migration of Jurkat cells and early endothelial progenitor cells was proven to demonstrate the applicability of the approach to endogenously CXCR4 expressing cell types. Thus, this work offers new options for enhancing chemotactic hCXCL12 gradients by a combination of native and modified hCXCL12 variants to improve and prolong the recruitment of CXCR4-positive stem and progenitor cells to injured sites.

Sulfated saccharides are an essential part of extracellular matrices, and they are involved in a large number of interactions. Sulfated saccharide matrices in organisms accumulate heavy metal ions in addition to other essential metal ions. Accumulation of heavy metal ions alters the function of the organisms and cells, resulting in severe and irreversible damage. The effect of the sulfation pattern of saccharides on heavy metal binding preferences is enigmatic because the accessibility to structurally defined sulfated saccharides is limited and because standard analytical techniques cannot be used to quantify these interactions. We developed a new strategy that combines enzymatic and chemical synthesis with surface chemistry and label-free electrochemical sensing to study the interactions between well-defined sulfated saccharides and heavy metal ions. By using these tools we showed that the sulfation pattern of hyaluronic acid governs their heavy metal ions binding preferences., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

More than 100 hydrophobicity scales have been introduced, with each being based on a distinct condensed-phase approach. However, a comparison of the hydrophobicity values gained from different techniques, and their relative ranking, is not straightforward, as the interactions between the environment and the amino acid are unique to each method. Here, we overcome this limitation by studying the properties of amino acids in the clean-room environment of the gas phase. In the gas phase, entropic contributions from the hydrophobic effect are by default absent and only the polarity of the side chain dictates the self-assembly. This allows for the derivation of a novel hydrophobicity scale, which is based solely on the interaction between individual amino acid units within the cluster and thus more accurately reflects the intrinsic nature of a side chain. This principle can be further applied to classify non-natural derivatives, as shown here for fluorinated amino acid variants., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Functional biomaterials that are able to bind, stabilize and release bioactive proteins in a defined manner are required for the controlled delivery of such to the desired place of action, stimulating wound healing in health-compromised patients. Glycosaminoglycans (GAG) represent a very promising group of components since they may be functionally engineered and are well tolerated by the recipient tissues due to their relative immunological inertness. Ligands of the Epidermal Growth Factor (EGF) receptor (EGFR) activate keratinocytes and dermal fibroblasts and, thus, contribute to skin wound healing. Heparin-binding EGF-like growth factor (HB-EGF) bound to GAG in biomaterials (e.g. hydrogels) might serve as a reservoir that induces prolonged activation of the EGF receptor and to recover disturbed wound healing. Based on previous findings, the capacity of hyaluronan (HA) and its sulfated derivatives (sHA) to bind and release HB-EGF from HA/collagen-based hydrogels was investigated. Docking and molecular dynamics analysis of a molecular model of HB-EGF led to the identification of residues in the heparin-binding domain of the protein being essential for the recognition of GAG derivatives. Furthermore, molecular modeling and surface plasmon resonance (SPR) analyses demonstrated that sulfation of HA increases binding strength to HB-EGF thus providing a rationale for the development of sHA-containing hydrogels. In line with computational observations and in agreement with SPR results, gels containing sHA displayed a retarded HB-EGF release in vitro compared to pure HA/collagen gels. Hydrogels containing HA and collagen or a mixture with sHA were shown to bind and release bioactive HB-EGF over at least 72 h, which induced keratinocyte migration, EGFR-signaling and HGF expression in dermal fibroblasts. Importantly, hydrogels containing sHA strongly increased the effectivity of HB-EGF in inducing epithelial tip growth in epithelial wounds shown in a porcine skin organ culture model. These findings suggest that hydrogels containing HA and sHA can be engineered for smart and effective wound dressings. STATEMENT OF SIGNIFICANCE: Immobilization and sustained release of recombinant proteins from functional biomaterials might overcome the limited success of direct application of non-protected solute growth factors during the treatment of impaired wound healing. We developed HA/collagen-based hydrogels supplemented with acrylated sulfated HA for binding and release of HB-EGF. We analyzed the molecular basis of HB-EGF interaction with HA and its chemical derivatives by in silico modeling and surface plasmon resonance. These hydrogels bind HB-EGF reversibly. Using different in vitro assays and organ culture we demonstrate that the introduction of sulfated HA into the hydrogels significantly increases the effectivity of HB-EGF action on target cells. Therefore, sulfated HA-containing hydrogels are promising functional biomaterials for the development of mediator releasing wound dressings., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Protein-templated fragment ligations have been established as a powerful method for the assembly and detection of optimized protein ligands. Initially developed for reversible ligations, the method has been expanded to irreversible reactions enabling the formation of super-additive fragment combinations. Here, protein-induced Mannich ligations are discovered as a biocatalytic reaction furnishing inhibitors of the transcription factor STAT5. STAT5 protein catalyzes multicomponent reactions of a phosphate mimetic, formaldehyde, and 1H-tetrazoles yielding protein ligands with greatly increased binding affinity and ligand efficiency. Reactions are induced under physiological conditions selectively by native STAT5 but not by other proteins. Formation of ligation products and (auto-)inhibition of the reaction are quantified and the mechanism is investigated. Inhibitors assembled by STAT5 block specifically the phosphorylation of this protein in a cellular model of acute myeloid leukemia (AML), DNA-binding of STAT5 dimers, expression of downstream targets of the transcription factor, and the proliferation of cancer cells in mice.

Binding of sulfated glycosaminoglycans (GAG) to a wide spectrum of extracellular regulatory proteins is crucial for physiological processes such as cell growth, migration, tissue homeostasis and repair. Thus, GAG derivatives exhibit great relevance in the development of innovative biomaterials for tissue regeneration therapies. We present a synthetic strategy for the preparation of libraries of defined sulfated oligohyaluronans as model GAG systematically varied in length, sulfation pattern and anomeric substitution in order to elucidate the effects of these parameters on GAG recognition by regulatory proteins. Through an experimental and computational approach using fluorescence polarization, ITC, docking and molecular dynamics simulations we investigate the binding of these functionalized GAG derivatives to ten representative regulatory proteins including IL-8, IL-10, BMP-2, sclerostin, TIMP-3, CXCL-12, TGF-β, FGF-1, FGF-2, and AT-III, and we establish structure-activity relationships for GAG recognition. Binding is mainly driven by enthalpy with only minor entropic contributions. In several cases binding is determined by GAG length, and in all cases by the position and number of sulfates. Affinities strongly depend on the anomeric modification of the GAG. Highest binding affinities are effected by anomeric functionalization with large fluorophores and by GAG dimerization. Our experimental and theoretical results suggest that the diversity of GAG binding sites and modes is responsible for the observed high affinities and other binding features. The presented new insights into GAG-protein recognition will be of relevance to guide the design of GAG derivatives with customized functions for the engineering of new biomaterials.

Polysialyltransferases synthesize polysialic acid on cell surface-expressed glycoconjugates, which is crucial for developing processes and signaling pathways in eukaryotes. Recent advances in cancer research have rendered polysialyltransferases important drug targets because polysialic acid contributes to cancer cell progression, metastasis, and treatment of resistant tumors. To aid the development of high-throughput screening assays for polysialyltransferase inhibitors, we demonstrate that a previously developed class of fluorescent CMP-sialic acid mimetics for sialyltransferases has nanomolar affinities for oligo- and polysialyltransferases and can be used for the rapid screening of new polysialyltransferase inhibitors. We demonstrate that these CMP-Neu5Ac mimetics inhibit polysialylation in vitro and perform cell culture experiments, where we observe reduced polysialylation of NCAM. Furthermore, we describe the structural basis of CMP-Neu5Ac mimetics binding to the human oligosialyltransferase ST8SiaIII and extrapolate why their affinity is high for human polysialyltransferases. Our results show that this novel class of compounds is a promising tool for the development of potent and selective drugs against polysialyltransferase activity.

Toll-like receptor 2 (TLR2) induces early inflammatory responses to pathogen and damage-associated molecular patterns trough heterodimerization with either TLR1 or TLR6. Since overstimulation of TLR2 signaling is linked to several inflammatory and metabolic diseases, TLR2 antagonists may provide therapeutic benefits for the control of inflammatory conditions. We present virtual screening for the identification of novel TLR2 modulators, which combines analyses of known ligand sets with structure-based approaches. The 13 identified compounds were pharmacologically characterized in HEK293-hTLR2 cells, THP-1 macrophages and peripheral blood mononuclear cells for their ability to inhibit TLR2-mediated responses. Four out of 13 selected compounds show concentration-dependent activity, representing a hit rate of 31%. The most active compound is the pyrogallol derivative MMG-11 that inhibits both TLR2/1 and TLR2/6 signaling and shows a higher potency than the previously discovered CU-CPT22. Concentration ratio analysis identified both compounds as competitive antagonists of Pam 3 CSK 4 - and Pam 2 CSK 4 -induced responses. Schild plot analysis yielded apparent pA 2 values of 5.73 and 6.15 (TLR2/1), and 5.80 and 6.65 (TLR2/6) for CU-CPT22 and MMG-11, respectively. MMG-11 neither shows cellular toxicity nor interference with signaling induced by other TLR agonists, IL-1β or TNF. Taken together, we demonstrate that MMG-11 is a potent and selective TLR2 antagonist with low cytotoxicity rendering it a promising pharmacological tool for the investigation of TLR signaling and a suitable lead structure for further chemical optimization., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Lead structure discovery mainly focuses on the identification of noncovalently binding ligands. Covalent linkage, however, is an essential binding mechanism for a multitude of successfully marketed drugs, although discovered by serendipity in most cases. We present a concept for the design of fragments covalently binding to proteases. Covalent linkage enables fragment binding unrelated to affinity to shallow protein binding sites and at the same time allows differentiated targeted hit verification and binding location verification through mass spectrometry. We describe a systematic and rational computational approach for the identification of covalently binding fragments from compound collections inhibiting enteroviral 3C protease, a target with high therapeutic potential. By implementing reactive groups potentially forming covalent bonds as a chemical feature in our 3D pharmacophore methodology, covalent binders were discovered by high-throughput virtual screening. We present careful experimental validation of the virtual hits using enzymatic assays and mass spectrometry unraveling a novel, previously unknown irreversible inhibition of the 3C protease by phenylthiomethyl ketone-based fragments. Subsequent synthetic optimization through fragment growing and reactivity analysis against catalytic and noncatalytic cysteines revealed specific irreversible 3C protease inhibition.

α,α-Difluoro-benzyl phosphonates are currently the most popular class of phosphotyrosine mimetics. Structurally derived from the natural substrate phosphotyrosine, they constitute classical bioisosteres and have enabled the development of potent inhibitors of protein tyrosine phosphatases (PTP) and phosphotyrosine recognition sites such as SH2 domains. Being dianions bearing two negative charges, phosphonates, however, do not permeate membranes and thus are often inactive in cells and have not been a successful starting point toward therapeutics, yet. In this work, benzyl phosphonates were modified by replacing phosphorus-bound oxygen atoms with phosphorus-bound fluorine atoms. Surprisingly, mono-P-fluorophosphonates were fully stable under physiological conditions, thus enabling the investigation of their mode of action toward PTP. Three alternative scenarios were tested and mono-P-fluorophosphonates were identified as stable reversible PTP1B inhibitors, despite of the loss of one negative charge and the replacement of one oxygen atom as an H-bond donor by fluorine. In extending this replacement strategy, α,α-difluorobenzyl penta-P-fluorophosphates were synthesized and found to be novel phosphotyrosine mimetics with improved affinity to the phosphotyrosine binding site of PTP1B., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background and Purpose: Gain of function mutations in TRPC6 channels can cause autosomal dominant forms of focal segmental glomerulosclerosis (FSGS). Validated inhibitors of TRPC6 channels that are biologically active on FSGS-related TRPC6 mutants are eagerly sought., Experimental Approach: We synthesized new TRPC6-inhibiting modulators from larixol, a resiniferous constituent of Larix decidua, and tested the potency and selectivity in cell lines stably expressing various TRPC channel isoforms. Channel activation was followed by Ca 2+ influx analyses and electrophysiological recordings. The most promising compound larixyl carbamate (LC) was tested on native TRPC6 channels and TRPC6 constructs carrying FSGS-related point mutations., Key Results: LC exhibited an about 30-fold preference for TRPC6 over TRPC3 channels and a fivefold preference for TRPC6 over TRPC7 channels. Six FSGS-related TRPC6 mutants, including the highly active M132T and R175Q variants, were strongly inhibited by 1 μM LC. Surprisingly, no TRPC6-related Ca 2+ signals were detectable in primary murine podocytes, or in acutely isolated glomeruli. in these preparations. Quantitative PCR revealed a 20-fold to 50-fold lower abundance of TRPC6 transcripts in rat or mouse podocytes, compared with pulmonary artery smooth muscle cells from the same species. Accordingly, electrophysiological recordings demonstrated that DAG-induced currents in murine podocytes are very small, but sensitive to LC., Conclusions and Implications: In spite of their low abundance in native podocytes, native TRPC6 channels are targetable using larixol-derived TRPC6 inhibitors. As observed with wild-type TRPC6 channels, FSGS-related TRPC6 mutants were sensitive to the newly developed inhibitors, paving the way for experimental therapies., (© 2017 The British Pharmacological Society.)

Reconstructed human epidermis (RHE) is used for risk assessment of chemicals and cosmetics and RHE as well as reconstructed human full-thickness skin (RHS) become important for e.g., the pre-clinical development of drugs. Yet, the knowledge regarding their biotransformation capacity is still limited, although the metabolic activity is highly relevant for skin sensitization, genotoxicity, and the efficacy of topical dermatics. The biotransformation of the aromatic amine 2,4-toluenediamine (2,4-TDA) has been compared in two commercially available RHS to normal human skin ex vivo, and in primary epidermal keratinocytes and dermal fibroblasts as well as in vitro generated epidermal Langerhans cells and dermal dendritic cells. The mono N-acetylated derivative N-(3-amino-4-methyl-phenyl)acetamide (M1) was the only metabolite detectable in substantial amounts indicating the predominance of N-acetylation. RHS exceeded human skin ex vivo in N-acetyltransferase activity and in cell cultures metabolite formation ranked as follows: keratinocytes > fibroblasts ~ Langerhans cells ~ dendritic cells. In conclusion, our results underline the principal suitability of RHS as an adequate test matrix for the investigation of N-acetylation of xenobiotics which is most relevant for risk assessment associated with cutaneous exposure to aromatic amines.

Bacterial biosurfactants are nature's strategy to solubilize and ingest hydrophobic molecules and nutrients using a fully biodegradable transport system. Eight structurally defined rhamnolipids were selected and investigated as potential drug carrier systems. Depending on the molecular structures defining their packing parameters, the rhamnolipids were found to form spherical nanoparticles with precisely defined average sizes between 5 and 100nm, low polydispersity, and stability over a broad concentration range as revealed from dynamic light scattering and electron microscopy. As rhamnolipids were tolerated well by the human skin, rhamnolipid nanoparticles were considered for dermal drug delivery and thus loaded with hydrophobic drug molecules. Using the drug model, Nile red, dexamethasone, and tacrolimus nanoparticles charged with up to 30% drug loading (w/w) were obtained. Nanoparticles loaded with Nile red were investigated for dermal drug delivery in a Franz cell using human skin. Fluoresence microscopy of skin slices indicated the efficient penetration of the model drug into human skin, both into the stratum corneum and although to a lesser extent into the lower epidermis. Rhamnolipid nanocarriers were found to be non-toxic to primary human fibroblasts in a proliferation assay and thus are considered candidates for the dermal delivery of drugs., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Competing Interests: Die Autoren erklären, dass keine Interessenkonflikte vorliegen.

Protein-templated fragment ligation is a novel concept to support drug discovery and can help to improve the efficacy of protein ligands. Protein-templated fragment ligations are chemical reactions between small molecules ("fragments") utilizing a protein's surface as a reaction vessel to catalyze the formation of a protein ligand with increased binding affinity. The approach exploits the molecular recognition of reactive small-molecule fragments by proteins both for ligand assembly and for the identification of bioactive fragment combinations. In this way, chemical synthesis and bioassay are integrated in one single step. This Review discusses the biophysical basis of reversible and irreversible fragment ligations and gives an overview of the available methods to detect protein-templated ligation products. The chemical scope and recent applications as well as future potential of the concept in drug discovery are reviewed., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Glycosaminoglycans are known to bind biological mediators thereby modulating their biological activity. Sulfated hyaluronans (sHA) were reported to strongly interact with transforming growth factor (TGF)-β1 leading to impaired bioactivity in fibroblasts. The underlying mechanism is not fully elucidated yet. Examining the interaction of all components of the TGF-β1:receptor complex with sHA by surface plasmon resonance, we could show that highly sulfated HA (sHA3) blocks binding of TGF-β1 to its TGF-β receptor-I (TβR-I) and -II (TβR-II). However, sequential addition of sHA3 to the TβR-II/TGF-β1 complex led to a significantly stronger recruitment of TβR-I compared to a complex lacking sHA3, indicating that the order of binding events is very important. Molecular modeling suggested a possible molecular mechanism in which sHA3 could potentially favor the association of TβR-I when added sequentially. For the first time bioactivity of TGF-β1 in conjunction with sHA was investigated at the receptor level. TβR-I and, furthermore, Smad2 phosphorylation were decreased in the presence of sHA3 indicating the formation of an inactive signaling complex. The results contribute to an improved understanding of the interference of sHA3 with TGF-β1:receptor complex formation and will help to further improve the design of functional biomaterials that interfere with TGF-β1-driven skin fibrosis.

Several pathologic conditions such as rheumatoid arthritis, ocular neovascularization, cancer, or atherosclerosis are often associated with abnormal angiogenesis, which requires innovative biomaterial-based treatment options to control the activity of angiogenic factors. Here, we studied how sulfated hyaluronan (sHA) and oversulfated chondroitin sulfate derivatives as potential components of functional biomaterials modulate vascular endothelial growth factor-A (VEGF-A) signaling and endothelial cell activity in vitro. Tissue inhibitor of metalloproteinase-3 (TIMP-3), an effective angiogenesis inhibitor, exerts its activity by competing with VEGF-A for binding to VEGF receptor-2 (VEGFR-2). However, even though TIMP-3 and VEGF-A are known to interact with glycosaminoglycans (GAGs), the potential role and mechanism by which GAGs alter the VEGF-A/TIMP-3 regulated VEGFR-2 signaling remains unclear. Combining surface plasmon resonance, immunobiochemical analysis, and molecular modeling, we demonstrate the simultaneous binding of VEGF-A and TIMP-3 to sHA-coated surfaces and identified a novel mechanism by which sulfated GAG derivatives control angiogenesis: GAG derivatives block the binding of VEGF-A and TIMP-3 to VEGFR-2 thereby reducing their biological activity in a defined, sulfation-dependent manner. This effect was stronger for sulfated GAG derivatives than for native GAGs. The simultaneous formation of TIMP-3/sHA complexes partially rescues the sHA inhibited VEGF-A/VEGFR-2 signaling and endothelial cell activation. These results provide novel insights into the regulation of angiogenic factors by GAG derivatives and highlight the potential of sHA derivatives for the treatment of diseases associated with increased VEGF-A and VEGFR-2 levels.

Protein-templated reactions enable the target-guided formation of protein ligands from reactive fragments, ideally with no background reaction. Herein, we investigate the templated formation of amides. A nucleophilic fragment that binds to the coagulation factor Xa was incubated with the protein and thirteen differentially activated dipeptides. The protein induced a non-catalytic templated reaction for the phenyl and trifluoroethyl esters; the latter was shown to be a completely background-free reaction. Starting from two fragments with millimolar affinity, a 29 nm superadditive inhibitor of factor Xa was obtained. The fragment ligation reaction was detected with high sensitivity by an enzyme activity assay and by mass spectrometry. The reaction progress and autoinhibition of the templated reaction by the formed ligation product were determined, and the structure of the protein-inhibitor complex was elucidated., (© 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Immune response against pathogens is a tightly regulated process that must ensure microbial control while preserving integrity of the infected organs. Tuberculosis (TB) is a paramount example of a chronic infection in which antimicrobial immunity is protective in the vast majority of infected individuals but can become detrimental if not finely tuned. Here, we report that C-type lectin dendritic cell (DC) immunoreceptor (DCIR), a key component in DC homeostasis, is required to modulate lung inflammation and bacterial burden in TB. DCIR is abundantly expressed in pulmonary lesions in Mycobacterium tuberculosis-infected nonhuman primates during both latent and active disease. In mice, we found that DCIR deficiency impairs STAT1-mediated type I IFN signaling in DCs, leading to increased production of IL-12 and increased differentiation of T lymphocytes toward Th1 during infection. As a consequence, DCIR-deficient mice control M. tuberculosis better than WT animals but also develop more inflammation characterized by an increased production of TNF and inducible NOS (iNOS) in the lungs. Altogether, our results reveal a pathway by which a C-type lectin modulates the equilibrium between infection-driven inflammation and pathogen's control through sustaining type I IFN signaling in DCs., Competing Interests: The authors declare no conflict of interest.

Chemical synthesis of oligosaccharide conjugates is essential for studying the functional relevance of carbohydrates, and this task would be facilitated considerably if reliable methods for the anomeric ligation of unprotected sugars in water were available. Here, a method for the preparation of anomeric glycosyl thiols from complex unprotected mono-, di-, and oligosaccharides is presented. By exploiting the neighboring-group effect of the 2-acetamido-group, 1,2-oxazolines are generated and converted into 1-glycosyl thioesters through treatment with 1-thioacids. The unprotected anomeric glycosyl thiolates released in situ were conjugated to Michael acceptors, aliphatic halogenides, and aziridines to furnish versatile glycoconjugates. Conjugation of amino acids and proteins was accomplished using the thiol-ene reaction with terminal olefins. This method gives efficient access to anomeric glycosyl thiols and thiolates, which enables anomeric ligations of complex unprotected glycans in water., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The stromal cell-derived factor 1α (CXCL12) belongs to the CXC chemokine family and plays an important role in tissue regeneration and the recruitment of stem cells. Here, a stable chemotactic gradient is essential that is formed by the interaction of CXCL12 with the extracellular matrix. Binding properties of CXCL12 to naturally occurring glycosaminoglycans (GAGs) as well as to the artificial highly sulfated hyaluronic acid (HA) are investigated by using a combination of NMR spectroscopy, molecular modeling and molecular dynamics simulations. Our results demonstrate a preferred protein binding for the sulfated GAGs heparin (HE) and highly sulfated HA. Furthermore, we could demonstrate that the orientation of the sulfate is crucial for binding. All sulfated GAGs interact with the CXCL12 GAG-binding motif (K24-H25-L26-K27-R41-K43-R47), where K27 and R41 represent the anchor points. Furthermore, differences could be observed in the second interaction interface of CXCL12: both HE and highly sulfated HA interfere with the receptor-binding motif, while chondroitin sulfate binds different amino acids in close proximity to this motif. CXCL12 does not interact with HA, which was directly demonstrated by NMR spectroscopy and molecular modeling and explained by the lack of sulfate groups of the HA molecule., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

An imbalance between tissue-degrading matrix metalloproteinases (MMPs) and their counterparts' tissue inhibitors of metalloproteinases (TIMPs) causes pathologic extracellular matrix (ECM) degradation in chronic wounds and requires new adaptive biomaterials that interact with these regulators to re-establish their balance. Sulfated glycosaminoglycans (GAGs) and TIMP-3 are key modulators of tissue formation and remodeling. However, little is known about their molecular interplay. GAG/TIMP-3 interactions were characterized combining surface plasmon resonance, ELISA, molecular modeling and hydrogen/deuterium exchange mass spectrometry. We demonstrate the potential of solute and surface-bound sulfated hyaluronan (sHA) and chondroitin sulfate (sCS) derivatives to manipulate GAG/TIMP-3 interactions by varying GAG concentration, sulfation degree and chain length. Three GAG binding sites in the N- and C-terminal domains of TIMP-3 were identified. We reveal no overlap with the matrix metalloproteinases (MMP)-binding site, elucidating why GAGs did not change MMP-1/-2 inhibition by TIMP-3 in enzyme kinetics. Since we prove that GAGs alone have a low impact on MMP activity, sHA and sCS offer a promising strategy to possibly control ECM remodeling via stabilizing and accumulating TIMP-3 by maintaining its MMP inhibitory activity under GAG-bound conditions. Whether GAG-based functional biomaterials can be applied to foster chronic wound healing by shifting the MMP/TIMP balance to a healing promoting state needs to be evaluated in vivo., Statement of Significance: Increased levels of tissue-degrading matrix metalloproteinases (MMPs) lead to pathologic matrix degradation in chronic wounds. Therefor functional biomaterials that restore the balance between MMPs and tissue inhibitors of metalloproteinases (TIMPs) are required to promote wound healing. Since sulfated glycosaminoglycan (GAG) derivatives demonstrated already to be e.g. anti-inflammatory and immunomodulatory, and native GAGs interact with TIMP-3 the former are promising candidates for functionalizing biomaterials. We identified the GAG binding sites of TIMP-3 by combining experimental and molecular modeling approaches and revealed that GAG derivatives have a higher capacity to sequester TIMP-3 than native GAGs without altering its inhibitory potential towards MMPs. Thus GAG derivative-containing biomaterials could protect tissue from excessive proteolytic degradation e.g. in chronic wounds by re-establishing the MMP/TIMP balance., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Sulfated glycosaminoglycans (sGAGs) modulate cellular processes via their interaction with extracellular matrix (ECM) proteins. We revealed a direct binding of tissue inhibitor of metalloproteinase-3 (TIMP-3) to the endocytic receptor low-density lipoprotein receptor-related protein (LRP-1) clusters II and IV using surface plasmon resonance. Sulfated hyaluronan (sHA) and chondroitin sulfate (sCS) derivatives interfered with TIMP-3/LRP-1 complex formation in a sulfation-dependent manner stronger than heparin. Electrostatic potential calculations suggested a competition between negatively charged GAGs and highly negatively charged complement-like domains of LRP-1 for the binding to a positively charged area of TIMP-3 as an underlying mechanism. In vitro studies revealed increased amounts of pericellular TIMP-3 in the presence of sHA as a consequence of the blocked protein uptake. GAG derivatives as part of biomaterials might post-translationally modulate TIMP-3 levels stronger than native GAGs, thus exhibiting catabolic effects on the ECM, which could prevent extensive pathological matrix degradation and promote wound healing.

Small-molecule fragments binding to biomacromolecules can be starting points for the development of drugs, but are often difficult to detect due to low affinities. Here we present a strategy that identifies protein-binding fragments through their potential to induce the target-guided formation of covalently bound, irreversible enzyme inhibitors. A protein-binding nucleophile reacts reversibly with a bis-electrophilic warhead, thereby positioning the second electrophile in close proximity of the active site of a viral protease, resulting in the covalent de-activation of the enzyme. The concept is implemented for Coxsackie virus B3 3C protease, a pharmacological target against enteroviral infections. Using an aldehyde-epoxide as bis-electrophile, active fragment combinations are validated through measuring the protein inactivation rate and by detecting covalent protein modification in mass spectrometry. The structure of one enzyme-inhibitor complex is determined by X-ray crystallography. The presented warhead activation assay provides potent non-peptidic, broad-spectrum inhibitors of enteroviral proteases.

Toxin-antitoxin systems constitute a native survival strategy of pathogenic bacteria and thus are potential targets of antibiotic drugs. Here, we target the Zeta-Epsilon toxin-antitoxin system, which is responsible for the stable maintenance of certain multiresistance plasmids in Gram-positive bacteria. Peptide ligands were designed on the basis of the ε₂ζ₂ complex. Three α helices of Zeta forming the protein-protein interaction (PPI) site were selected and peptides were designed conserving the residues interacting with Epsilon antitoxin while substituting residues binding intramolecularly to other parts of Zeta. Designed peptides were synthesized with an N-terminal fluoresceinyl-carboxy-residue for binding assays and provided active ligands, which were used to define the hot spots of the ε₂ζ₂ complex. Further shortening and modification of the binding peptides provided ligands with affinities <100 nM, allowing us to determine the most relevant PPIs and implement a robust competition binding assay.

Implants and artificial biomaterials containing sulfated hyaluronans have been shown to improve the healing of injured skin and bones. It is hypothesized that these effects are mediated by the binding of sulfated glycosaminoglycans (GAGs) to growth factors and cytokines, resulting in the sequestering of proteins to the wound healing site and in modulated protein activity. Given that no direct synthetic access to sulfated oligohyaluronans has been available, little is known about their protein binding and the structure of the resulting protein complexes. Here, the chemoenzymatic preparation of oligohyaluronans on the gram scale is described. Oligohyaluronans are converted into anomeric azides at the reducing end, enabling the attachment of analytical labels through an anomeric ligation reaction. A nonasulfated tetrahyaluronan-ethylenediaminetetraacetic acid derivative has been produced and used as a paramagnetic tag for the elucidation of the complex of this ligand with interleukin-10 using paramagnetic relaxation enhancement NMR analysis. The metal ion position is resolved with 1.0 Å, enabling a refined structural model of the complex., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cluster of differentiation 1c (CD1c)-dependent self-reactive T cells are abundant in human blood, but self-antigens presented by CD1c to the T-cell receptors of these cells are poorly understood. Here we present a crystal structure of CD1c determined at 2.4 Å revealing an extended ligand binding potential of the antigen groove and a substantially different conformation compared with known CD1c structures. Computational simulations exploring different occupancy states of the groove reenacted these different CD1c conformations and suggested cholesteryl esters (CE) and acylated steryl glycosides (ASG) as new ligand classes for CD1c. Confirming this, we show that binding of CE and ASG to CD1c enables the binding of human CD1c self-reactive T-cell receptors. Hence, human CD1c adopts different conformations dependent on ligand occupancy of its groove, with CE and ASG stabilizing CD1c conformations that provide a footprint for binding of CD1c self-reactive T-cell receptors.

The increasing need for site-specific protein decorations that mimic natural posttranslational modifications requires access to a variety of noncanonical amino acids with moieties enabling bioorthogonal conjugation chemistry. Here we present the incorporation of long-chain olefinic amino acids into model proteins with rational variants of pyrrolysyl-tRNA synthetase (PylRS). Nε-heptenoyl lysine was incorporated for the first time using the known promiscuous variant PylRS(Y306A/Y384F), and Nε-pentenoyl lysine was incorporated in significant yields with the novel variant PylRS(C348A/Y384F). This is the only example of rational modification at position C348 to enlarge the enzyme's binding pocket. Furthermore, we demonstrate the feasibility of our chosen amino acids in the thiol-ene conjugation reaction with a thiolated polysaccharide.

The biological function of interleukin-10 (IL-10), a pleiotropic cytokine with an essential role in inflammatory processes, is known to be affected by glycosaminoglycans (GAGs). GAGs are highly negatively charged polysaccharides and integral components of the extracellular matrix with important functions in the biology of many growth factors and cytokines. The molecular mechanism of the IL-10/GAG interaction is unclear. In particular, experimental evidence about IL-10/GAG binding sites is lacking, despite its importance for understanding the biological role of the interaction. Here, we report the experimental determination of a GAG binding site of IL-10. Although no co-crystal structure of the IL-10·GAG complex could be obtained, its structural characterization was possible by NMR spectroscopy. Chemical shift perturbations of IL-10 induced by GAG binding were used to narrow down the location of the binding site and to assess the affinity for different GAG molecules. Subsequent observation of NMR pseudocontact shifts of IL-10 and its heparin ligand, as induced by a protein-attached lanthanide spin label, provided structural restraints for the protein·ligand complex. Using these restraints, pseudocontact shift-based rigid body docking together with molecular dynamics simulations yielded a GAG binding model. The heparin binding site is located at the C-terminal end of helix D and the adjacent DE loop and coincides with a patch of positively charged residues involving arginines 102, 104, 106, and 107 and lysines 117 and 119. This study represents the first experimental characterization of the IL-10·GAG complex structure and provides the starting point for revealing the biological significance of the interaction of IL-10 with GAGs., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Classical or canonical transient receptor potential 6 (TRPC6), a nonselective and Ca(2+)-permeable cation channel, mediates pathophysiological responses within pulmonary and renal diseases that are still poorly controlled by current medication. Thus, controlling TRPC6 activity may provide a promising and challenging pharmacological approach. Recently identified chemical entities have demonstrated that TRPC6 is pharmacologically targetable. However, isotype-selectivity with regard to its closest relative, TRPC3, is difficult to achieve. Reasoning that balsams, essential oils, or incense materials that are traditionally used for inhalation may contain biologic activities to block TRPC6 activity, we embarked on a natural compound strategy to identify new TRPC6-blocking chemical entities. Within several preparations of plant extracts, a strong TRPC6-inhibitory activity was found in conifer balsams. The biologic activity was associated with nonvolatile resins, but not with essential oils. Of various conifers, the larch balsam was unique in displaying a marked TRPC6-prevalent mode of action. By testing the main constituents of larch resin, we identified larixol and larixyl acetate as blockers of Ca(2+) entry and ionic currents through diacylglycerol- or receptor-activated recombinant TRPC6 channels, exhibiting approximately 12- and 5-fold selectivity compared with its closest relatives TRPC3 and TRPC7, respectively. No significant inhibition of more distantly related TRPV or TRPM channels was seen. The potent inhibition of recombinant TRPC6 by larixyl acetate (IC50 = 0.1-0.6 µM) was confirmed for native TRPC6-like [Ca(2+)]i signals in diacylglycerol-stimulated rat pulmonary artery smooth muscle cells. In isolated mouse lungs, larix-6-yl monoacetate (CAS 4608-49-5; larixyl acetate; 5 µM) prevented acute hypoxia-induced vasoconstriction. We conclude that larch-derived labdane-type diterpenes are TRPC6-selective inhibitors and may represent a starting point for pharmacological TRPC6 modulation within experimental therapies., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Phosphopeptide mimetics containing the 4-phosphonocarbonyl phenylalanine (pcF) as a photo-active phosphotyrosine isoster are developed as potent, light-switchable inhibitors of the protein tyrosine phosphatase PTP1B. The photo-active inhibitors 6-10 are derived from phosphopeptide substrates and are prepared from the suitably protected pcF building block 12 by Fmoc-based solid phase peptide synthesis. All pcF-containing peptides are moderate inhibitors of PTP1B with KI values between 10 and 50μM. Irradiation of the inhibitors at 365nm in the presence of the protein PTP1B amplify the inhibitory activity of pcF-peptides up to 120-fold, switching the KI values of the best inhibitors to the sub-micromolar range. Photo-activation of the inhibitors results in the formation of triplet intermediates of the benzoylphosphonate moiety, which deactivate PTP1B following an oxidative radical mechanism. Deactivation of PTP1B proceeds without covalent crosslinking of the protein target with the photo-switched inhibitors and can be reverted by subsequent addition of reducing agent dithiothreitol (DTT)., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In this study, we have used techniques from cell biology, biochemistry, and genetics to investigate the role of the tyrosine phosphatase Shp2 in tumor cells of MMTV-PyMT mouse mammary glands. Genetic ablation or pharmacological inhibition of Shp2 induces senescence, as determined by the activation of senescence-associated β-gal (SA-β-gal), cyclin-dependent kinase inhibitor 1B (p27), p53, and histone 3 trimethylated lysine 9 (H3K9me3). Senescence induction leads to the inhibition of self-renewal of tumor cells and blockage of tumor formation and growth. A signaling cascade was identified that acts downstream of Shp2 to counter senescence: Src, focal adhesion kinase, and Map kinase inhibit senescence by activating the expression of S-phase kinase-associated protein 2 (Skp2), Aurora kinase A (Aurka), and the Notch ligand Delta-like 1 (Dll1), which block p27 and p53. Remarkably, the expression of Shp2 and of selected target genes predicts human breast cancer outcome. We conclude that therapies, which rely on senescence induction by inhibiting Shp2 or controlling its target gene products, may be useful in blocking breast cancer., (© 2015 The Authors.)

Three polymers, poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA), hyperbranched polyglycerol (hPG), and dextran were investigated as carriers for multivalent ligands targeting the adaptive tandem WW-domain of formin-binding protein (FBP21). Polymer carriers were conjugated with 3-9 copies of the proline-rich decapeptide GPPPRGPPPR-NH2 (P1). Binding of the obtained peptide-polymer conjugates to the tandem WW-domain was investigated employing isothermal titration calorimetry (ITC) to determine the binding affinity, the enthalpic and entropic contributions to free binding energy, and the stoichiometry of binding for all peptide-polymer conjugates. Binding affinities of all multivalent ligands were in the µM range, strongly amplified compared to the monovalent ligand P1 with a K D > 1 mM. In addition, concise differences were observed, pHPMA and hPG carriers showed moderate affinity and bound 2.3-2.8 peptides per protein binding site resulting in the formation of aggregates. Dextran-based conjugates displayed affinities down to 1.2 µM, forming complexes with low stoichiometry, and no precipitation. Experimental results were compared with parameters obtained from molecular dynamics simulations in order to understand the observed differences between the three carrier materials. In summary, the more rigid and condensed peptide-polymer conjugates based on the dextran scaffold seem to be superior to induce multivalent binding and to increase affinity, while the more flexible and dendritic polymers, pHPMA and hPG are suitable to induce crosslinking upon binding.

Selective inhibitors of the protein tyrosine phosphatase SHP2 (src homology region 2 domain phosphatase; PTPN11), an enzyme that is deregulated in numerous human tumors, were generated through a combination of chemical synthesis and structure-based rational design. Seventy pyridazolon-4-ylidenehydrazinyl benzenesulfonates were prepared and evaluated in enzyme assays. The binding modes of active inhibitors were simulated in silico using a newly generated crystal structure of SHP2. The most powerful compound, GS-493 (4-{(2Z)-2-[1,3-bis(4-nitrophenyl)-5-oxo-1,5-dihydro-4H-pyrazol-4-yliden]hydrazino}benzenesulfonic acid; 25) inhibited SHP2 with an IC50 value of 71±15 nM in the enzyme assay and was 29- and 45-fold more active toward SHP2 than against related SHP1 and PTP1B. In cell culture experiments compound 25 was found to block hepatocyte growth factor (HGF)-stimulated epithelial-mesenchymal transition of human pancreatic adenocarcinoma (HPAF) cells, as indicated by a decrease in the minimum neighbor distances of cells. Moreover, 25 inhibited cell colony formation in the non-small-cell lung cancer cell line LXFA 526L in soft agar. Finally, 25 was observed to inhibit tumor growth in a murine xenograft model. Therefore, the novel specific compound 25 strengthens the hypothesis that SHP2 is a relevant protein target for the inhibition of mobility and invasiveness of cancer cells., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Arginine-rich cell-penetrating peptides (CPP) are widely employed as delivery vehicles for a large variety of macromolecular cargos. As a mechanism-of-action for induction of uptake cross-linking of heparan sulfates and interaction with lipid head groups have been proposed. Here, we employed a multivalent display of the CPP nona-arginine (R9) on a linear dextran scaffold to assess the impact of heparan sulfate and lipid interactions on uptake and membrane perturbation. Increased avidity through multivalency should potentiate molecular phenomena that may only play a minor role if only individual peptides are used. To this point, the impact of multivalency has only been explored for dendrimers, CPP-decorated proteins and nanoparticles. We reasoned that multivalency on a linear scaffold would more faithfully mimic the arrangement of peptides at the membrane at high local peptide concentrations. On average, five R9 were coupled to a linear dextran backbone. The conjugate displayed a direct cytoplasmic uptake similar to free R9 at concentrations higher than 10μM. However, this uptake was accompanied by an increased membrane disturbance and cellular toxicity that was independent of the presence of heparan sulfates. In contrast, for erythrocytes, the multivalent conjugate induced aggregation, however, showed only limited membrane perturbation. Overall, the results demonstrate that multivalency of R9 on a linear scaffold strongly increases the capacity to interact with the plasma membrane. However, the induction of membrane perturbation is a function of the cellular response to peptide binding., (Copyright © 2014. Published by Elsevier B.V.)