70 research outputs found
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 proteaseâfragmentâpre-
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 1â3 and S1-binding fragments 4â17. 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 Abstractâą Referencesâą Author
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Author information Abstractâą Referencesâą Author informationâą Supplementary
information Affiliations Institute of Pharmacy, Medicinal Chemistry,
University of Leipzig, BrĂŒderstraĂe 34, 04103 Leipzig, Germany Edyta Burda &
Jörg Rademann Institute of Pharmacy, Medicinal Chemistry, Freie UniversitÀt
Berlin, Königin-Luise-StraĂe 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 Abstractâą Referencesâą Author informationâą
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
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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 proteaseâfragmentâpre-
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
PeptideâBismuth 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. Peptideâbismuth 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 150â
nM, respectively. Bicyclic peptides are up to 130â
times more active and 19â
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 150â
nM, respectively. Bicyclic peptides are up to 130â
times more active and 19â
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 cmâ1 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-ÎČ1 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)-ÎČ1 leading to impaired
bioactivity in fibroblasts. The underlying mechanism is not fully elucidated
yet. Examining the interaction of all components of the TGF-ÎČ1:receptor
complex with sHA by surface plasmon resonance, we could show that highly
sulfated HA (sHA3) blocks binding of TGF-ÎČ1 to its TGF-ÎČ receptor-I (TÎČR-I)
and -II (TÎČR-II). However, sequential addition of sHA3 to the TÎČR-II/TGF-ÎČ1
complex led to a significantly stronger recruitment of TÎČR-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 TÎČR-I when added
sequentially. For the first time bioactivity of TGF-ÎČ1 in conjunction with sHA
was investigated at the receptor level. TÎČR-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-ÎČ1:receptor
complex formation and will help to further improve the design of functional
biomaterials that interfere with TGF-ÎČ1-driven skin fibrosis
Peptideâmediated surface coatings for the release of woundâhealing 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 1.8â
ÎŒM at a molecular weight of 211â
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 structureâactivity relationships of chemically stable TLR2 antagonists
Tollâlike receptors (TLRs) build the first barrier in the innate immune response and therefore represent promising targets for the modulation of inflammatory processes. Recently, the pyrogallolâcontaining TLR2 antagonists CUâCPT22 and MMGâ11 were reported; however, their 1,2,3âtriphenol 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 â9 ) based on the systematic variation of substructures, linker elements, and the hydrogenâbonding pattern of the pyrogallol precursors by using chemically robust building blocks. The novel series of chemically stable and synthetically accessible TLR2 antagonists (1 â9 ) was pharmacologically characterized, and the potential binding modes of the active compounds were evaluated structurally. Our results provide new insights into structureâactivity 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, TLR2âselective, and shows a similar activity with regard to the pyrogallol starting points, thus indicating the variability of the hydrogen bonding pattern
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