Catalytic activation of pre-substrates via dynamic Fragment assembly on Protein templates

Sensitive detection of small molecule fragments binding to defined sites of biomacromolecules is still a considerable challenge. Here we demonstrate that protein-binding fragments are able to induce enzymatic reactions on the protein surface via dynamic fragment ligation. Fragments binding to the S1 pocket of serine proteases containing a nitrogen, oxygen or sulphur nucleophile are found to activate electrophilic pre-substrates through a reversible, covalent ligation reaction. The dynamic ligation reaction positions the pre-substrate molecule at the active site of the protein thereby inducing its enzymatic cleavage. Catalytic activation of pre-substrates is confirmed by fluorescence spectroscopy and by high-performance liquid chromatography. The approach is investigated with 3 pre-substrates and 14 protein-binding fragments and the specific activation and the templating effect exerted by the enzyme is quantified for each protease–fragment–pre- substrate combination. The described approach enables the site-specific identification of protein-binding fragments, the functional characterization of enzymatic sites and the quantitative analysis of protein template-assisted ligation reactions. View full text Subject terms: Chemical sciences Chemical biology Medicinal chemistry At a glance Figures First | 1-4 of 6 | Last View all figures left The concept of pre-substrate activation by protein-binding fragments. Figure 1 Proof-of-concept. Figure 2 Structure of potential pre- substrates 1–3 and S1-binding fragments 4–17. Figure 3 Model for the activation of pre-substrates by nucleophilic protein-binding fragments. Figure 4 Activation of pre-substrate 3. Figure 5 Three-fragment assembly. Figure 6 right Compounds Genes and Proteins References Abstract• References• Author information• Supplementary information Rees, D. C., Congreve, M., Murray, C. W. & Carr, R. Fragment-based lead discovery. Nat. Rev. Drug Discov. 3, 660–672 (2004). 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CAS ISI Article Download references Author information Abstract• References• Author information• Supplementary information Affiliations Institute of Pharmacy, Medicinal Chemistry, University of Leipzig, Brüderstraße 34, 04103 Leipzig, Germany Edyta Burda & Jörg Rademann Institute of Pharmacy, Medicinal Chemistry, Freie Universität Berlin, Königin-Luise-Straße 2+4, 14195 Berlin, Germany Jörg Rademann Contributions J.R. and E.B. conceived and designed the experiments, E.B. performed the experiments. Both authors discussed the results and co-wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: Jörg Rademann Supplementary information Abstract• References• Author information• Supplementary information PDF files Supplementary Information (915 KB) Supplementary Figures 1-11, Supplementary Methods and Supplementary References. Additional data 3-Oxo-(N-(4-methyl-2-oxo-2H- chromen-7-yl)-propanoylamide N-(4-Methyl-2-oxo-2H-chromen-7-yl)-acrylamide 3-((2-Aminoethyl)thio)-N-(4-methyl-2-oxo-2H-chromen-7-yl)propanamide npj Genomic Medicine - Open for Submissions Science jobs Science events NatureEvents Directory The 1st Annual Translational Microbiome Conference 14 May 2015 — 15 May 2015 Boston, MA, United States rTMS for depression, OCD and new developments: 2-day rTMS course 19 November 2015 — 20 November 2015 Bijleveldsingel 34, Nijmegen, Netherlands Human Health in the Face of Climate Change: Science, Medicine, and Adaptation 14 May 2015 — 15 May 2015 Carrer d' Isaac Newton, 26, Barcelona, Spain Post a free event More science events Discover more LIF negatively regulates tumour-suppressor p53 through Stat3/ID1/MDM2 in colorectal cancers Nature Communications 17 Oct 2014 Interlocked loops trigger lineage specification and stable fates in the Drosophila nervous system Nature Communications 28 Jul 2014 O-GlcNAc- modification of SNAP-29 regulates autophagosome maturation Nature Cell Biology 24 Nov 2014 Most read Nature.com Open innovation Pavillion Intravenous Sustained Release Drug Delivery Technology Deadline: Mar 21 2015 Reward: $30,000 USD Intravenous delivery allows for the rapid distribution of injected drugs from the veins throughout the entire body. Oftentim… Wireless Internet Connectivity for Field Applications Deadline: Apr 05 2015 Reward:$20,000 USD Data collection in outdoor field studies is problematic and inefficient without a reliable wireless internet connection. The… Powered by:innocentive Sensitive detection of small molecule fragments binding to defined sites of biomacromolecules is still a considerable challenge. Here we demonstrate that protein-binding fragments are able to induce enzymatic reactions on the protein surface via dynamic fragment ligation. Fragments binding to the S1 pocket of serine proteases containing a nitrogen, oxygen or sulphur nucleophile are found to activate electrophilic pre-substrates through a reversible, covalent ligation reaction. The dynamic ligation reaction positions the pre-substrate molecule at the active site of the protein thereby inducing its enzymatic cleavage. Catalytic activation of pre-substrates is confirmed by fluorescence spectroscopy and by high-performance liquid chromatography. The approach is investigated with 3 pre-substrates and 14 protein-binding fragments and the specific activation and the templating effect exerted by the enzyme is quantified for each protease–fragment–pre- substrate combination. The described approach enables the site-specific identification of protein-binding fragments, the functional characterization of enzymatic sites and the quantitative analysis of protein template-assisted ligation reactions.1\. Auflag

Characterization of defined sulfated heparin-like oligosaccharides by electrospray ionization ion trap mass spectrometry

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

Peptide–Bismuth Bicycles: In Situ Access to Stable Constrained Peptides with Superior Bioactivity

Constrained peptides are promising next-generation therapeutics. We report here a fundamentally new strategy for the facile generation of bicyclic peptides using linear precursor peptides with three cysteine residues and a non-toxic trivalent bismuth(III) salt. Peptide–bismuth bicycles form instantaneously at physiological pH, are stable in aqueous solution for many weeks, and much more resistant to proteolysis than their linear precursors. The strategy allows the in situ generation of bicyclic ligands for biochemical screening assays. We demonstrate this for two screening campaigns targeting the proteases from Zika and West Nile viruses, revealing a new lead compound that displayed inhibition constants of 23 and 150 nM, respectively. Bicyclic peptides are up to 130 times more active and 19 times more proteolytically stable than their linear analogs without bismuth.C.N. thanks the Australian Research Council for funding (DE190100015 and DP200100348). The work was supported by the DFG-funded Core Facility BioSupraMol. We thank Prof. Gottfried Otting for help with NMR spectroscopy, Dr. Josemon George for sample preparation, Dr. Christoph Arkona for plasmid transformation, Silke Bergemann for support with protein expression and purification, and Peter Demirel for providing a sample of Fmoc-Phe(4-Boc2-guanidino)-OH, development of the HPLC method, and MS support

Peptide-Bismuth Bicycles: In Situ Access to Stable Constrained Peptides with Superior Bioactivity

Constrained peptides are promising next-generation therapeutics. We report here a fundamentally new strategy for the facile generation of bicyclic peptides using linear precursor peptides with three cysteine residues and a non-toxic trivalent bismuth(III) salt. Peptide-bismuth bicycles form instantaneously at physiological pH, are stable in aqueous solution for many weeks, and much more resistant to proteolysis than their linear precursors. The strategy allows the in situ generation of bicyclic ligands for biochemical screening assays. We demonstrate this for two screening campaigns targeting the proteases from Zika and West Nile viruses, revealing a new lead compound that displayed inhibition constants of 23 and 150 nM, respectively. Bicyclic peptides are up to 130 times more active and 19 times more proteolytically stable than their linear analogs without bismuth.C.N. thanks the Australian Research Council for funding (DE190100015 and DP200100348). The work was supported by the DFG-funded Core Facility BioSupraMol. We thank Prof. Gottfried Otting for help with NMR spectroscopy, Dr. Josemon George for sample preparation, Dr. Christoph Arkona for plasmid transformation, Silke Bergemann for support with protein expression and purification, and Peter Demirel for providing a sample of Fmoc-Phe(4-Boc2- guanidino)-OH, development of the HPLC method, and MS support

IR action spectroscopy of glycosaminoglycan oligosaccharides

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

Reliable palladium nanoparticle syntheses in aqueous solution: the importance of understanding precursor chemistry and growth mechanism

Reliable protocols for the synthesis of palladium nanoparticles (Pd-NPs) in aqueous solution are rarely found and the corresponding growth mechanisms often remain unknown. Furthermore, syntheses of Pd-NPs always demand the use of stabilizing agents which are often unfavorable for catalytic applications. In this contribution, the importance of the palladium precursor chemistry as a prerequisite for any reliable Pd-NP synthesis in aqueous solution is shown. This includes a detailed study of the influence of the precursor chemistry on the nanoparticle growth mechanism. The findings enable the controlled modification of a common synthetic protocol (i.e. the reduction of a palladium precursor with NaBH4) to obtain sub-5 nm Pd-NPs without the use of any stabilizing agent. In addition, it is also shown that such mechanistic studies are not only of great importance to the development of novel synthetic procedures. Exemplarily, the successful transfer of the synthesis from lab-to large-scale is demonstrated.BMBF, 03EK3009, Design hocheffizienter Elektrolysekatalysatore

Possible Consequences for TGF-β1 Signaling

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

Peptide‐mediated surface coatings for the release of wound‐healing cytokines

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

A Critical Study on Acylating and Covalent Reversible Fragment Inhibitors of SARS-CoV-2 Main Protease Targeting the S1 Site with Pyridine

SARS coronavirus main proteases (3CL proteases) have been validated as pharmacological targets for the treatment of coronavirus infections. Current inhibitors of SARS main protease, including the clinically admitted drug nirmatrelvir are peptidomimetics with the downsides of this class of drugs including limited oral bioavailability, cellular permeability, and rapid metabolic degradation. Here, we investigate covalent fragment inhibitors of SARS Mpro as potential alternatives to peptidomimetic inhibitors in use today. Starting from inhibitors acylating the enzyme's active site, a set of reactive fragments was synthesized, and the inhibitory potency was correlated with the chemical stability of the inhibitors and the kinetic stability of the covalent enzyme-inhibitor complex. We found that all tested acylating carboxylates, several of them published prominently, were hydrolyzed in assay buffer and the inhibitory acyl-enzyme complexes were rapidly degraded leading to the irreversible inactivation of these drugs. Acylating carbonates were found to be more stable than acylating carboxylates, however, were inactive in infected cells. Finally, reversibly covalent fragments were investigated as chemically stable SARS CoV-2 inhibitors. Best was a pyridine-aldehyde fragment with an IC50 of 1.8 μM at a molecular weight of 211 g/mol, showing that pyridine fragments indeed are able to block the active site of SARS-CoV-2 main protease

Biological characterization, mechanistic investigation and structure‐activity relationships of chemically stable TLR2 antagonists

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