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). CAS ISI PubMed Article Hajduk, P. J. & Greer, J. A decade of fragment- based drug design: strategic advances and lessons learned. Nat. Rev. Drug Discov. 6, 211–219 (2007). CAS ISI PubMed Article Erlanson, D. A., McDowell, R. S. & O’Brien, T. Fragment-based drug discovery. J. Med. Chem. 47, 3463–3482 (2004). CAS ISI PubMed Article Fattori, D., Squarcia, A. & Bartoli, S. Fragment-based approach to drug lead discovery. Drugs RD 9, 217–227 (2008). ISI Article Rademann, J. Organic protein chemistry: drug discovery through the chemical modification of proteins. Angew. Chem. Int. Ed. 43, 4554–4556 (2004). Article Corbett, P. T. et al. Dynamic combinatorial chemistry. Chem. Rev. 106, 3652–3711 (2006). CAS ISI PubMed Article Erlanson, D. A. Fragment-based lead discovery: a chemical update. Curr. Opin. Biotechnol. 17, 643–652 (2006). CAS ISI PubMed Article Schmidt, M. F. & Rademann, J. Dynamic template-assisted strategies in fragment-based drug discovery. Trends Biotechnol. 27, 512–521 (2009). CAS ISI PubMed Article Shuker, S. B., Hajduk, P. J., Meadows, R. P. & Fesik, S. W. Discovering high-affinity ligands for proteins: SAR by NMR. Science 274, 1531–1534 (1996). CAS ISI PubMed Article Vongvilai, P., Angelin, M., Larsson, R. & Ramström, O. Dynamic combinatorial resolution: direct asymmetric lipase-mediated screening of dynamic nitroaldol library. Angew. Chem. Int. Ed. 46, 948–950 (2007). CAS Article Nienaber, V. L. et al. Discovering novel ligands for macromolecules using X-ray crystallographic screening. Nat. Biotechnol. 18, 1105–1108 (2000). CAS ISI PubMed Article Blundell, T. L., Jhoti, H. & Abell, C. High-throughput crystallography for lead discovery in drug design. Nat. Rev. Drug Discov. 1, 45–54 (2002). CAS ISI PubMed Article Sharff, A. & Jhoti, H. High-throughput crystallographyto enhance drug discovery. Curr. Opin. Chem. Biol. 7, 340–345 (2003). CAS ISI PubMed Article Sanders, W. J. et al. Discovery of potent inhibitors of dihydroneopterin aldolase using CrystaLEAD high-throughput X-ray crystallographic screening and structure-directed lead optimization. J. Med. Chem. 47, 1709–1718 (2004). CAS ISI PubMed Article Patterson, A. W., Wood, W. J. L. & Ellman, J. A. Substrate activity screening (SAS): a general procedure for the preparation and screening of a fragment-based non-peptidic protease substrate library for inhibitor discovery. Nat. Protoc. 2, 424–433 (2007). ISI PubMed Article Mares-Guia, M. & Shaw, E. Substrate side chain guanidines as model of the binding of amidines and studies on the active center of trypsin. J. Biol. Chem. 240, 1579–1585 (1965). CAS PubMed Schmidt, M. F. et al. Sensitized detection of inhibitory fragments and iterative development of non- peptidic protease inhibitors by dynamic ligation screening. Angew. Chem. Int. Ed. 47, 3275–3278 (2008). Article Schmidt, M. F., El-Dahshan, A., Keller, S. & Rademann, J. Selective identification of cooperatively binding fragments in a high-throughput ligation assay enables development of a picomolar caspase-3 inhibitor. Angew. Chem. Int. Ed. 48, 6346–6349 (2009). CAS Article Schmidt, M. F., Groves, M. & Rademann, J. Dynamic substrate enhancement for the identification of specific, second-site-binding fragments targeting a set of protein tyrosine phosphatases. Chembiochem 12, 2640–2646 (2011). ISI PubMed Article Al-Gharabli, S. I. et al. An efficient method for the synthesis of peptide aldehyde libraries employed in the discovery of reversible SARS coronavirus main protease (SARS-CoV Mpro) inhibitors. Chembiochem 7, 1048–1055 (2006). ISI PubMed Article Reek J. N. H., Otto S. (eds)Dynamic Combinatorial Chemistry Wiley-VCH (2010). Osowska, K. & Miljanic, O. S. Oxidative kinetic self-sorting of a dynamic imine library. J. Am. Chem. Soc. 133, 724–727 (2011). ISI PubMed Article Hermann, A. Dynamic mixtures and combinatorial libraries: imines as probes for molecular evolution at the interface between chemistry and biology. Org. Biomol. Chem. 7, 3195–3204 (2009). PubMed Article Meyer, C. D., Joiner, C. S. & Stoddart, J. F. Template-directed synthesis employing reversible imine bond formation. Chem. Soc. Rev. 36, 1705–1723 (2007). CAS ISI PubMed Article Rodriguez-Decampo, Z. & Otto, S. Orthogonal or simultaneous use of disulfide and hydrazone exchange in dynamic covalent chemistry in aqueous solution. Chem. Commun. 5301–5303 (2008). Dirksen, A., Yegneswaran, S. & Dawson, P. E. Bisaryl hydrazones as exchangeable biocompatible linkers. Angew. Chem. Int. Ed. 49, 2023–2027 (2010). CAS Article Soutullo, M. D., O’Brien, R. A., Gaines, K. E. & Davis, J. H. Jr Constitutional dynamic systems of ionic and molecular liquids. Chem. Commun. 2529–2531 (2009). Drahonovsky, D. & Lehn, J.-M. Hemiacetals in dynamic covalent chemistry: formation, exchange, selection and modulation processes. J. Org. Chem. 74, 8428–8432 (2009). ISI PubMed Article Caraballo, R., Dong, H., Ribeiro, J. P., Jimenez-Barbero, J. & Ramström, O. Direct STD NMR identification of β-galactosidase inhibitors from a virtual dynamic hemithioacetal system. Angew. Chem. Int. Ed. 49, 589–593 (2010). Article Cacciapaglia, R., Di Stefano, S. & Mandolini, L. Metathesis reaction of formaldehyde acetals: an easy entry into the dynamic covalent chemistry of cyclophane formation. J. Am. Chem. Soc. 127, 13666–13671 (2005). ISI PubMed Article Berkovich-Berger, D. & Lemcoff, N. G. Facile acetal dynamic combinatorial library. Chem. Commun. 1686–1688 (2008). Larsson, R., Pei, Z. & Ramström, O. Catalytic self-screening of cholinesterase substrates from a dynamic combinatorial thioester library. Angew. Chem. Int. Ed. 43, 3716–3718 (2004). Article Shi, B. & Greaney, M. F. Reversible Michael addition of thiols as a new tool for dynamic combinatorial chemistry. Chem. Commun. 886–888 (2005). de Candia, M., Lopopolo, G. & Altomare, C. Novel factor Xa inhibitors: a patent review. Expert Opin. Ther. Pat. 19, 1535–1580 (2009). PubMed Article Schröder Leiros, H.-H. et al. Trypsin specificity as elucidated by LIE calculations, X-ray structures, and association constant measurements. Protein Sci. 13, 1056–1070 (2004). PubMed Article List, B., Lerner, R. A. & Barbas, C. F. III Proline-catalyzed direct asymmetric aldol reactions. J. Am. Chem. Soc. 122, 2395–2396 (2000). CAS ISI Article Wilhelms, N., Kuchat, S. & Lehn, J.-M. Organocatalysis of C=N/C=N and C=C/C=N exchange in dynamic covalent chemistry. Helv. Chim. Acta 95, 2635–2651 (2012). ISI Article Khatik, G. L., Kumar, R. & Chakraborti, A. K. Catalyst-free conjugate addition of thiols to α,β-unsaturated carbonyl compounds in water. Org. Lett. 8, 2433–2436 (2006). PubMed Article Hoyle, C. E. & Bowman, C. N. Thiol-ene click chemistry. Angew. Chem. Int. Ed. 49, 1540–1573 (2010). 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

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

Graphene Oxide alpha Bi2O3 Composites for Visible Light Photocatalysis, Chemical Catalysis and Solar Energy Conversion

The growing challenges of environmental purification by solar photocatalysis, precious metal free catalysis and photocurrent generation in photovoltaic cells are receiving the utmost global attention. Here we demonstrate the one pot green chemical synthesis of a new stable heterostructured, eco friendly, multifunctional micro composite consisting of amp; 945; Bi2O3 micro needles intercalated with anchored graphene oxide GO micro sheets 1.0 wt for the above mentioned applications in a large economical scale. The bare amp; 945; Bi2O3 micro needles display twice as better photocatalytic activities than commercial TiO2 Degussa P25 while the GO hybridized composite exhibit 4 6 times enhanced photocatalytic activities than neat TiO2 photocatalyst in the degradation of colored aromatic organic dyes crystal violet and rhodamine 6G under visible light irradiation 300 W tungsten lamp . The highly efficient activity is associated with the strong surface adsorption ability of GO for aromatic dye molecules, the high carrier acceptability and efficient electron hole pair separation in Bi2O3 by individual adjoining GO sheets. Introduction of Ag nanoparticles 2.0 wt further enhances the photocatalytic performance of the composite over 8 folds due to a plasmon induced electron transfer process from Ag nanoparticles via GO sheets into the conduction band of Bi2O3. The new composites are also catalytically active. They catalyze the reduction of 4 nitrophenol to 4 aminophenol in presence of borohydride ions. Photoanodes assembled from GO amp; 945; Bi2O3 and Ag GO amp; 945; Bi2O3 composites display an improved photocurrent response power conversion efficiency 20 higher over those prepared without GO in dye sensitized solar cells DSSCs

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

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

Nonuniform friction-area dependency for antimony oxide surfaces sliding on graphite

Cataloged from PDF version of article.We present frictional measurements involving controlled lateral manipulation of antimony nanoparticles on graphite featuring atomically smooth particle-substrate interfaces via tapping- and contact-mode atomic force microscopy. As expected from earlier studies, the power required for lateral manipulation as well as the frictional forces recorded during the manipulation events exhibit a linear dependence on the contact area over a wide size range from 2000 nm2 to 120 000 nm2. However, we observe a significant and abrupt increase in frictional force and dissipated power per contact area at a value of about 20 000 nm2, coinciding with a phase transition from amorphous to crystalline within the antimony particles. Our results suggest that variations in the structural arrangement and stoichiometry of antimony oxide at the interface between the particles and the substrate may be responsible for the observed effect. © 2013 American Physical Society

On Track or Off The Rails?

In order to achieve a transition from a transport system centred on the individual car to one centred on (electrified) rail a new focus in infrastructure planning is needed. The preparation of project proposals for the Federal Transport Infrastructure Plan 2030 on the sub-national level in Germany provides an opportunity to study decision-making processes in ministries and compare their respective results in this respect. Using document analysis, expert interviews, qualitative content analysis as well as QCA, this thesis in political science analyses how decision-making processes within bureaucracies impact the decision output in transport infrastructure planning. It contributes to the discussion on bureaucracy-politics interactions that is relevant beyond the German case. One result is that ministries tend to use complex decision-making processes for topics deemed salient as long as the available capacity permits it. Consequently, in order to conduct legitimacy-enhancing steps – such as public participation – a well-funded bureaucracy is indispensable

Irreversible inhibitors of the 3C protease of Coxsackie virus through templated assembly of protein-binding fragments

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

Mitochondria-Targeted Antioxidants SkQ1 and MitoTEMPO Failed to Exert a Long-Term Beneficial Effect in Murine Polymicrobial Sepsis

Mitochondrial-derived reactive oxygen species have been deemed an important contributor in sepsis pathogenesis. We investigated whether two mitochondria-targeted antioxidants (mtAOX; SkQ1 and MitoTEMPO) improved long-term outcome, lessened inflammation, and improved organ homeostasis in polymicrobial murine sepsis. 3-month-old female CD-1 mice (n = 90) underwent cecal ligation and puncture (CLP) and received SkQ1 (5 nmol/kg), MitoTEMPO (50 nmol/kg), or vehicle 5 times post-CLP. Separately, 52 SkQ1-treated CLP mice were sacrificed at 24 h and 48 h for additional endpoints. Neither MitoTEMPO nor SkQ1 exerted any protracted survival benefit. Conversely, SkQ1 exacerbated 28-day mortality by 29%. CLP induced release of 10 circulating cytokines, increased urea, ALT, and LDH, and decreased glucose but irrespectively of treatment. Similar occurred for CLP-induced lymphopenia/neutrophilia and the NO blood release. At 48 h post-CLP, dying mice had approximately 100-fold more CFUs in the spleen than survivors, but this was not SkQ1 related. At 48 h, macrophage and granulocyte counts increased in the peritoneal lavage but irrespectively of SkQ1. Similarly, hepatic mitophagy was not altered by SkQ1 at 24 h. The absence of survival benefit of mtAOX may be due to the extended treatment and/or a relatively moderate-risk-of-death CLP cohort. Long-term effect of mtAOX in abdominal sepsis appears different to sepsis/inflammation models arising from other body compartments

Phase diagram of the extended Hubbard chain with charge-dipole interactions

We consider a modified extended Hubbard model (EHM) which, in addition to the on-site repulsion U and nearest-neighbor repulsion V, includes polarization effects in second-order perturbation theory. The model is equivalent to an EHM with renormalized U plus a next-nearest-neighbor repulsion term. Using a method based on topological quantum numbers (charge and spin Berry phases), we generalize to finite hopping t the quantum phase diagram in one dimension constructed by van den Brink et al. (Phys. Rev. Lett. 75, 4658 (1995)). At hopping t=0 there are two charge density-wave phases, one spin density-wave phase and one intermediate phase with charge and spin ordering, depending on the parameter values. At t \neq 0 the nature of each phase is confirmed by studying correlation functions. However, in addition to the strong-coupling phases, a small region with bond ordering appears. The region occupied by the intermediate phase first increases and then decreases with increasing t, until it finally disappears for t of the order but larger than U. For small t, the topological transitions agree with the results of second order perturbation theory.Comment: 6 pages, 5 figures, two columns latex version. Accepted for publication in Physical Review B. Mistaken reference 16 has been correcte