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

Characterisation of ten NS2B-NS3 proteases: Paving the way for pan-flavivirus drugs

Flaviviruses can cause severe illness in humans. Effective and safe vaccines are available for some species; however, for many flaviviruses disease prevention or specific treatments remain unavailable. The viral replication cycle depends on the proteolytic activity of the NS2B-NS3 protease, which releases functional viral proteins from a non-functional polyprotein precursor, rendering the protease a promising drug target. In this study, we characterised recombinant NS2B-NS3 proteases from ten flaviviruses including three unreported proteases from the Usutu, Kyasanur forest disease and Powassan viruses. All protease constructs comprise a covalent Gly4-Ser-Gly4 linker connecting the NS3 serine protease domain with its cofactor NS2B. We conducted a comprehensive cleavage site analysis revealing areas of high conversion. While all proteases were active in enzymatic assays, we noted a 1000-fold difference in catalytic efficiency across proteases from different flaviviruses. Two bicyclic peptide inhibitors displayed anti-pan-flaviviral protease activity with inhibition constants ranging from 10 to 1000 nM

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

Protein-Templated Ugi Reactions versus In-Situ Ligation Screening: Two Roads to the Identification of SARS-CoV-2 Main Protease Inhibitors

Protein-templated fragment ligation was established as a method for the rapid identification of high affinity ligands, and multicomponent reactions (MCR) such as the Ugi four-component reaction (Ugi 4CR) have been efficient in the synthesis of drug candidates. Thus, the combination of both strategies should provide a powerful approach to drug discovery. Here, we investigate protein-templated Ugi 4CR quantitatively using a fluorescence-based enzyme assay, HPLC-QTOF mass spectrometry (MS), and native protein MS with SARS-CoV-2 main protease as template. Ugi reactions were analyzed in aqueous buffer at varying pH and fragment concentration. Potent inhibitors of the protease were formed in presence of the protein via Ugi 4CR together with Ugi three-component reaction (Ugi 3CR) products. Binding of inhibitors to the protease was confirmed by native MS and resulted in the dimerization of the protein target. Formation of Ugi products was, however, more efficient in the non-templated reaction, apparently due to interactions of the protein with the isocyanide and imine fragments. Consequently, in-situ ligation screening of Ugi 4CR products was identified as a superior approach to the discovery of SARS-CoV-2 protease inhibitors

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

Dimerization and crowding in the binding of interleukin 8 to dendritic glycosaminoglycans as artificial proteoglycans

Interactions of glycosaminoglycans (GAG) with proteins of the extracellular matrix govern and regulate complex physiological functions including cellular growth, immune response, and inflammation. Repetitive presentation of GAG binding motifs as found in native proteoglycans might enhance GAG–protein binding through multivalent interactions. Here, we report the chemical synthesis of dendritic GAG oligomers constructed of nona-sulfated hyaluronan tetrasaccharides for investigating the binding of the protein chemokine interleukin 8 (IL-8) to artificial, well-defined proteoglycan architectures. Binding of mutant monomeric and native dimerizable IL-8 was investigated by nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry. Dendritic oligomerization of GAG increased the binding affinity of both monomeric and dimeric IL-8. Monomeric IL‑8 bound to monomeric and dimeric GAG with KD values of 7.3 µM and 0.108 µM, respectively. The effect was less pronounced for dimerizable wildtype IL-8, for which GAG dimerization improved the affinity from 34 nM to 5 nM. Binding of dimeric IL-8 to oligomeric GAG was limited by steric crowding effects, strongly reducing the affinity of subsequent binding events. In conclusion, the strongest effect of GAG oligomerization was the amplified binding of IL-8 monomers, which might concentrate monomeric protein in the extracellular matrix and thus promote protein dimerization under physiological conditions