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Journal Cover Sosyoloji Konferansları (Istanbul Journal of Sociological Studies)
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  This is an Open Access Journal Open Access journal
   ISSN (Print) 1304-0243
   Published by İstanbul Üniversitesi Homepage  [10 journals]
  • Different Enzymatic Processing of gamma Phosphoramidate and gamma
           Phosphoester modified ATP Analogs

    • Authors: Andreas Marx; Susanne Ermert, Stephan Hacker, Martin Scheffner, Christof Hauck, Alexander Buntru
      Abstract: Monitoring the activity of ATP-consuming enzymes provides the basis for elucidating their modes of action and regulation. Although a number of modified ATP analogs have been developed for this purpose, their scope is still restricted due to limited acceptance by respective enzymes. To clarify which kind of phosphate modified ATP analogs are accepted by the alpha-beta phosphoanhydride cleaving ubiquitin activating enzyme 1 (UBA1) and the beta-gamma phosphoanhydride cleaving focal adhesion kinase (FAK), we tested phosphoramidate and phosphoester modified ATP analogs. We report that UBA1 and FAK are able to convert phosphoramidate modified ATP analogs, even with a bulky modification like biotin. In contrast, a respective phosphoester modified ATP analog is poorly accepted. These results demonstrate that minor variations in the design of ATP analogs for monitoring ATP utilization have a significant impact on their enzymatic acceptance.
      PubDate: 2016-12-09T05:45:22.559747-05:
      DOI: 10.1002/cbic.201600590
       
  • Natural and Synthetic Macrocyclic Inhibitors of the Histone Deacetylase
           Enzymes

    • Authors: Alex R. Maolanon; Helle M. E. Kristensen, Luke J. Leman, M. Reza Ghadiri, Christian A. Olsen
      Abstract: Inhibition of histone deacetylase (HDAC) enzymes has emerged as a target for development of cancer chemotherapy. Four compounds have gained approval for clinical use by the Food and Drug Administration in the US, and several are currently in clinical trials. However, none of these compounds possesses particularly good isozyme selectivity, which would be a highly desirable feature in a tool compound. Whether selective inhibition of individual HDAC isozymes will provide improved drug candidates remains to be seen. Nevertheless, it has been speculated that using macrocyclic compounds to target HDAC enzymes might hold an advantage over the use of traditional hydroxamic-acid-containing inhibitors, which rely on chelation to the conserved active-site zinc ion. Here we review the literature on macrocyclic HDAC inhibitors obtained from natural sources and on structure–activity relationship studies inspired by these molecules, as well as on efforts aimed at fully synthetic macrocyclic HDAC inhibitors.Epigenetic drugs: Macrocyclic inhibitors of histone deacetylase (HDAC) enzymes have played important roles as tool compounds, including in the first isolation of mammalian HDACs, and one of a handful HDAC inhibitors approved for clinical use is a macrocyclic depsipeptide. In this article the chemistry and structure–activity relationships of macrocyclic HDAC inhibitors are comprehensively reviewed.
      PubDate: 2016-12-08T12:45:51.626716-05:
      DOI: 10.1002/cbic.201600519
       
  • Isolation and Characterization of RNA Aptamers against a
           Proteasome-Associated Deubiquitylating Enzyme UCH37

    • Authors: Jung Hoon Lee; Min Jae Lee
      Abstract: Deubiquitylating (DUB) enzymes antagonize ubiquitin-dependent protein degradation both before and after the substrates are engaged with proteasomes. UCH37 is one of three proteasome-associated DUB enzymes in mammals and the only protease among them from the ubiquitin carboxyl-terminal hydrolase (UCH) family. Here, we report the identification of specific RNA aptamers for UCH37 through in vitro selection, and we describe their inhibitory effects on the DUB activity of UCH37. The RNA aptamers significantly delayed RPN13-mediated UCH37 activation and lowered total DUB activity of proteasomes, as measured by the hydrolysis of ubiquitin-rhodamine 110. In addition, the UCH37 aptamers efficiently facilitated the hydrolysis of peptide-based reporter substrates of proteasomes. Thus, the UCH37 aptamers might offer a possible strategy for removing toxic cellular proteins through enhancing proteasome activity.Careful selection: Specific RNA aptamers of UCH37 were identified through an in vitro SELEX selection process. The most promising candidate inhibited deubiquitylation by UCH37 and activated proteasomal degradation, as measured by the hydrolysis of ubiquitin-rhodamine 110 and reporter peptide substrates.
      PubDate: 2016-12-08T12:45:44.778934-05:
      DOI: 10.1002/cbic.201600515
       
  • Brominated Luciferins Are Versatile Bioluminescent Probes

    • Authors: Rachel C. Steinhardt; Colin M. Rathbun, Brandon T. Krull, Jason M. Yu, Yuhang Yang, Brian D. Nguyen, Jake Kwon, David C. McCutcheon, Krysten A. Jones, Filipp Furche, Jennifer A. Prescher
      Abstract: We report a set of brominated luciferins for bioluminescence imaging. These regioisomeric scaffolds were accessed by using a common synthetic route. All analogues produced light with firefly luciferase, although varying levels of emission were observed. Differences in photon output were analyzed by computation and photophysical measurements. The brightest brominated luciferin was further evaluated in cell and animal models. At low doses, the analogue outperformed the native substrate in cells. The remaining luciferins, although weak emitters with firefly luciferase, were inherently capable of light production and thus potential substrates for orthogonal mutant enzymes.Bright ideas! Multicellular imaging and other applications of bioluminescence technology require diverse luciferin architectures. Here, we report a set of brominated luciferins that are robust light emitters and exhibit unique photophysical properties. These molecules can also be diversified by cross-coupling reactions to access an assortment of imaging probes.
      PubDate: 2016-12-08T12:45:28.70363-05:0
      DOI: 10.1002/cbic.201600564
       
  • The View from the Back Row

    • Authors: Meghan A. Campbell
      Abstract: Taking it all in: Exploring the role of editors at conferences will help you understand how to make the most of such an interaction. After wrapping up a successful 2016 conference season, the Editor-in-Chief explains the importance of these visits. Also included, is a summary of Volume 17 and a preview of the coming year.
      PubDate: 2016-12-08T12:45:22.481765-05:
      DOI: 10.1002/cbic.201600645
      Issue No: Vol. 17 (2016)
       
  • Identification of N-Arylated NH125 Analogues as Rapid Eradicating Agents
           against MRSA Persister Cells and Potent Biofilm Killers of Gram-Positive
           Pathogens

    • Authors: Yasmeen Abouelhassan; Akash Basak, Hussain Yousaf, Robert William Huigens III
      Abstract: Bacterial biofilms housing dormant persister cells are innately tolerant to antibiotics and disinfectants, yet several membrane-active agents are known to eradicate tolerant bacterial cells. NH125, a membrane-active persister-killer and starting point for development, led to the identification of N-arylated analogues 1 and 2 and displayed improved biofilm-killing potencies and rapid persister-killing activities in stationary cultures of methicillin-resistant Staphylococcus aureus (MRSA). We found 1 and 2 to be superior to other membrane-active agents in biofilm eradication assays with 1 demonstrating minimum biofilm eradication concentrations (MBEC) of 23.5, 11.7 and 2.35 µM against MRSA, methicillin-resistant S. epidermidis (MRSE) and vancomycin-resistant Enterococcus faecium (VRE) biofilms, respectively. We tested our panel of membrane-active agents against MRSA stationary cultures and found 1 to rapidly eradicate MRSA stationary cells by 4-logs (99.99%) in 30 minutes. The potent biofilm eradication and rapid persister cell killing exhibited by N-arylated NH125 analogues could have significant impact addressing biofilm-associated problems.
      PubDate: 2016-12-07T10:20:33.028918-05:
      DOI: 10.1002/cbic.201600622
       
  • A Method to Generate and Analyze Modified Myristoylated Proteins

    • Authors: Huanyao Gao; Wei Sun, Zhiquan Song, Yanbao Yu, Li Wang, Xian Chen, Qisheng Zhang
      Abstract: Covalent lipid modification of proteins is essential to their cellular localizations and functions. Engineered lipid motif coupled with bio-orthogonal chemistry has been utilized to identify myristoylated or palmitoylated proteins in cells. However, whether modified proteins have similar properties as endogenous ones has not been well investigated mainly due to lack of methods to generate and analyze purified proteins. We have developed a method that utilizes metabolic interference and mass spectrometry to produce and analyze modified, myristoylated small GTPase ADP-ribosylation factor 1 (Arf1). The capacities of these recombinant proteins to bind liposomes and load and hydrolyze GTP are measured and compared with the non-modified myristoylated Arf1. The ketone-modified myristoylated Arf1 can be further labeled by fluorophore-coupled hydrazine and subsequently visualized through fluorescence imaging. This methodology provides an effective model system to characterize lipid-modified proteins with additional functions before applying them to cellular systems.
      PubDate: 2016-12-07T10:20:31.992019-05:
      DOI: 10.1002/cbic.201600608
       
  • Efficient Conjugation of Oligosaccharides to Polymer Particles through
           Furan/Maleimide Diels–Alder Reaction: Application to the Capture of
           Carbohydrate-Binding Proteins

    • Authors: Antoine Petrelli; Eric Samain, Stéphanie Pradeau, Sami Halila, Sébastien Fort
      Abstract: Glycan–protein interactions play a crucial role in physiological and pathological events. Hence, improving the isolation of carbohydrate-binding proteins (i.e., lectins and anti-glycan antibodies) from complex media might not only lead to a better understanding of their function, but also provide solutions for public health issues, such as water contamination or the need for universal blood plasma. Here we report a rapid and efficient method for producing carbohydrate-based affinity adsorbents combining enzymatic synthesis and metal-free click chemistry. Both simple and complex glycans (maltose, blood group antigens A, B, and H) were readily modified by the addition of a furyl group at the reducing end without the need for protecting groups and were then efficiently conjugated to maleimide-activated Sepharose particles through Diels–Alder cycloaddition. These neoglycoconjugates showed high efficiency for the purification of lectins (concanavalin A and Ulex europaeus agglutinin), as well as for the capture of anti-A and anti-B blood group antibodies, opening new prospects for glycoproteomics and for the development of universal blood plasma.Fishing for carbohydrate-binding proteins: A protocol for the chemoselective modification of oligosaccharides and their conjugation through Diels–Alder cycloaddition has been developed. Carbohydrate affinity adsorbents were efficiently produced and used for the purification of lectins and for the capture of anti-blood-group antibodies.
      PubDate: 2016-12-07T07:42:04.324373-05:
      DOI: 10.1002/cbic.201600509
       
  • K-BILDS: A Kinase Substrate Discovery Tool

    • Authors: D. Maheeka Embogama; Mary Kay H. Pflum
      Abstract: Kinases catalyze protein phosphorylation to regulate cell signaling events. However, identifying kinase substrates is challenging due to the often low abundance and dynamic nature of protein phosphorylation. Development of novel techniques to identify kinase substrates is necessary. Here, we report kinase-catalyzed biotinylation with inactivated lysates for discovery of substrates (K-BILDS) as a tool to identify direct substrates of a kinase. As a proof of concept, K-BILDS was applied to cAMP-dependent protein kinase A (PKA) with HeLa cell lysates. Subsequent enrichment and MS/MS analysis identified 279 candidate PKA substrates, including 56 previously known PKA substrates. Of the candidate substrates, nuclear autoantigenic sperm protein (NASP), BCL2-associated athanogene 3 (BAG3), and 14-3-3 protein Tau (YWHAQ) were validated as novel PKA substrates. K-BILDS provides a valuable tool to identify direct substrates of any protein kinase.A tool that BILDS: Kinase substrate identification represents a significant challenge in cell signaling research. Kinase-catalyzed biotinylation with inactivated lysates for discovery of substrates (K-BILDS) offers a simple, yet powerful, method for detecting kinase substrate pairs.
      PubDate: 2016-12-07T07:41:59.375324-05:
      DOI: 10.1002/cbic.201600511
       
  • Corrigendum: A Tandem Green–Red Heterodimeric Fluorescent Protein
           with High FRET Efficiency

    • Authors: Matthew D. Wiens; Yi Shen, Xi Li, M. Alaraby Salem, Nick Smisdom, Wei Zhang, Alex Brown, Robert E. Campbell
      PubDate: 2016-12-07T07:41:30.827922-05:
      DOI: 10.1002/cbic.201600638
       
  • Optimization of Caged Electrophiles for Improved Monitoring of Cysteine
           Reactivity in Living Cells

    • Authors: Masahiro Abo; Daniel W. Bak, Eranthie Weerapana
      Abstract: Cysteine residues play critical roles in protein function and are susceptible to numerous post-translational modifications (PTMs) that serve to modulate the activity and localization of diverse proteins. Many of these PTMs are highly transient and labile, thus necessitating methods to study these modifications directly within the context of living cells. We previously reported a caged electrophilic probe, CBK1, that can be activated by UV for temporally controlled covalent modification of cysteine residues in living cells. To improve upon the number of cysteine residues identified in cellular cysteine-profiling studies, the reactivity and uncaging efficiency of a panel of caged electrophiles were explored. We identified an optimized caged electrophilic probe, CIK4, that affords significantly improved coverage of cellular cysteine residues. The broader proteome coverage afforded by CIK4 renders it a useful tool for the biological investigation of cysteine-reactivity changes and PTMs directly within living cells and highlights design elements that are critical to optimizing photoactivatable chemical probes for cellular labeling.I spy: Monitoring cysteine reactivity directly in living cells allows the elucidation of the sites of cysteine modification by oxidants as well as endogenous and exogenous thiol-reactive agents. We evaluated a panel of caged electrophiles as live-cell compatible cysteine-reactive probes and report an optimized probe for enhanced labeling of cellular cysteines.
      PubDate: 2016-12-05T05:20:49.806823-05:
      DOI: 10.1002/cbic.201600524
       
  • Effect of glycosylation on the biocatalytic properties of hydroxynitrile
           lyase from the passion fruit, Passiflora edulis - a comparison of natural
           and recombinant enzymes

    • Authors: Aem Nuylert; Yuko Ishida, Yasuhisa Asano
      Abstract: A hydroxynitrile lyase from the passion fruit, Passiflora edulis (PeHNL) was isolated from the leaves and showed high stability in biphasic co-organic solvent systems for cyanohydrin synthesis. Cyanohydrins are important building blocks for the production of fine chemicals and pharmaceuticals. Thus, to enhance production yields of PeHNL for industrial applications, we cloned and expressed recombinant PeHNL in E. coli BL21 (DE3) and P. pastoris GS115 cells without a signal peptide sequence. The aim of the present study is to determine the effect of N-glycosylation on enzyme stability and catalytic properties in microbial expression systems. PeHNL from leaves (PeHNL-N) and P. pastoris (PeHNL-P) was glycosylated, whereas that expressed in E. coli (PeHNL-E) was not. The enzymes PeHNL-N and PeHNL-P showed much better thermostability, pH stability, and organic solvent tolerance than the deglycosylated enzyme PeHNL-E and the deglycosylated mutant N105Q from P. pastoris (PeHNL-P-N105Q). The glycosylated PeHNL-P also efficiently performed transcyanation of (R)-mandelonitrile with a 98% enantiomeric excess in a biphasic system with 2-isopropyl ether. These data demonstrate the efficacy of the present methods for improving enzyme expression and stability for industrial application based on N-glycosylation.
      PubDate: 2016-12-03T03:50:28.843141-05:
      DOI: 10.1002/cbic.201600447
       
  • Asymmetric Ketone Reduction by Imine Reductases

    • Authors: Maike Lenz; Jan Meisner, Leann Quertinmont, Stefan Lutz, Johannes Kästner, Bettina Nestl
      Abstract: The rapidly growing area of asymmetric imine reduction by imine reductases (IREDs) has provided alternative routes to chiral amines. Here we report the expansion of the reaction scope of IREDs by showing the stereoselective reduction of 2,2,2-trifluoroacetophenone (1). Assisted by an in silico analysis of energy barriers, we evaluated asymmetric hydrogenations of carbonyls and imines considering the influence of substrate reactivity on the chemoselectivity of this novel class of reductases. We report on the asymmetric reduction of C=N as well as C=O bonds catalyzed by members of the IRED enzyme family.
      PubDate: 2016-12-02T12:09:43.36199-05:0
      DOI: 10.1002/cbic.201600647
       
  • Dendrimeric Guanidinoneomycin for Cellular Delivery of Bio-macromolecules

    • Authors: Aurora Sganappa; Ezequiel Wexselblatt, Maria Cristina Bellucci, Jeffrey D. Esko, Gabriella Tedeschi, Yitzhak Tor, Alessandro Volonterio
      Abstract: We present the synthesis of polymeric amino- and guanidinoglycosides prepared by tethering neomycin and guanidinoneomycin to PAMAM dendrimers of generations 2 and 4. The ability of these conjugates to promote cellular uptake of high-molecular-weight cargo is discussed, together with their cytotoxicity and mechanisms of entry. We demonstrate that the presence of multiple guanidinoneomycin carriers on the PAMAM surface plays an important role in promoting cellular uptake of the dendrimers, maintaining the heparan sulfate specificity and negligible cytotoxicity typical of monomeric guanidinoglycoside molecular transporters.Polymeric guanidinoglycoside vehicles: Comparing the cellular uptake of undecorated PAMAM dendrimers with that of PAMAM-neomycin and -guanidinoneomycin conjugates shows that the presence of guanidinoneomycins, to a greater extent than that of neomycins, plays an important role in promoting cellular uptake of high-molecular-weight cargos.
      PubDate: 2016-12-02T03:10:38.826689-05:
      DOI: 10.1002/cbic.201600422
       
  • Phylogenetic Studies, Gene Cluster Analysis, and Enzymatic Reaction
           Support Anthrahydroquinone Reduction as the Physiological Function of
           Fungal 17β-Hydroxysteroid Dehydrogenase

    • Authors: Leon Fürtges; David Conradt, Michael A. Schätzle, Shailesh Kumar Singh, Nada Kraševec, Tea Lanišnik Rižner, Michael Müller, Syed Masood Husain
      Abstract: 17β-Hydroxysteroid dehydrogenase (17β-HSDcl) from the filamentous fungus Curvularia lunata (teleomorph Cochliobolus lunatus) catalyzes NADP(H)-dependent oxidoreductions of androgens and estrogens. Despite detailed biochemical and structural characterization of 17β-HSDcl, its physiological function remains unknown. On the basis of amino acid sequence alignment, phylogenetic studies, and the recent identification of the physiological substrates of the homologous MdpC from Aspergillus nidulans and AflM from Aspergillus parasiticus, we propose an anthrahydroquinone as the physiological substrate of 17β-HSDcl. This is also supported by our analysis of a secondary metabolite biosynthetic gene cluster in C. lunata m118, containing 17β-HSDcl and ten other genes, including a polyketide synthase probably involved in emodin formation. Chemoenzymatic reduction of emodin by 17β-HSDcl in the presence of sodium dithionite verified this hypothesis. On the basis of these results, the involvement of a 17β-HSDcl in the biosynthesis of other anthrahydroquinone-derived natural products is proposed; hence, 17β-HSDcl should be more appropriately referred to as a polyhydroxyanthracene reductase (PHAR).Not a common transformation: The physiological function of 17β-hydroxysteroid dehydrogenase from the filamentous fungus Curvularia lunata (teleomorph Cochliobolus lunatus) is proposed as being an NADPH-dependent reduction of a polyhydroxyanthracene. With a substrate range from steroids to polyhydroxynaphthalenes, it might be seen as part of a diverse biosynthetic matrix.
      PubDate: 2016-12-01T09:47:26.181205-05:
      DOI: 10.1002/cbic.201600489
       
  • Improving the solubility of artificial ligands of streptavidin to enable
           more practical reversible switching of protein localization in cells

    • Authors: Ryo Tachibana; Takuya Terai, Gaelle Boncompain, Shigeru Sugiyama, Nae Saito, Franck Perez, Yasuteru Urano
      Abstract: Chemical inducers that can control target protein localization in living cells are powerful tools to investigate dynamic biological systems. We recently reported the "retention using selective hook" (RUSH) system for reversible localization change of proteins of interest by addition/washout of small molecules called artificial ligands of streptavidin (ALiS). However, the utility of previously developed ALiS is restricted by limited solubility in water. Here, we overcome this problem by X-ray crystal structure-guided design of a more soluble ALiS derivative (ALiS-3), which retains sufficient streptavidin-binding affinity for use in the RUSH system. The ALiS-3-streptavidin interaction was characterized in detail. We confirmed that ALiS-3 is a convenient and effective tool for dynamic control of α-mannosidase II localization between endoplasmic reticulum and Golgi in living cells.
      PubDate: 2016-11-30T23:16:52.814672-05:
      DOI: 10.1002/cbic.201600640
       
  • Fungal glycolipid hydrolase inhibitors and their effect on Cryptococcus
           neoformans

    • Authors: Andres Gonzalez Santana; Christina Tysoe, Guanggan Hu, James Kronstad, Ethan Goddard-Borger, Stephen Withers
      Abstract: Pathogenic fungi kill an estimated 1.3 million people each year. This number is predicted to rise as drug resistance spreads, thus antifungal drugs with novel modes of action are urgently required. Fungal endoglycoceramidase-related proteins 1 and 2 (EGCrP-1 and -2), which hydrolyse glucosylceramide and ergosteryl beta-glucoside, respectively, are important for fungal cell growth and have been identified as potential targets for drug development. A library of iminosugar derivatives was screened against EGCrP-1 and -2, thereby identifying a number of competitive inhibitors with nanomolar affinities. In addition, a mechanism-based inhibitor was shown to form a covalent derivative with EGCrP-2. Nine of the inhibitors were evaluated against Cryptococcus neoformans. Several showed growth inhibitory activity, but only against the C. neoformans strain lacking the outer fungal polysaccharide capsule, implying that penetration into the cell is a significant handicap for these inhibitors.
      PubDate: 2016-11-30T23:01:27.90056-05:0
      DOI: 10.1002/cbic.201600538
       
  • Zirconium (IV) Catalyzed Ring-Opening of On-DNA Epoxides in Water

    • Authors: Lijun Fan; Christopher P Davie
      Abstract: DNA-encoded library technology (ELT) has spurred wide interest in the pharmaceutical industry as a powerful tool for hit and lead generation. In recent years a number of "DNA-compatible" chemical modifications have been published and used to synthesize vastly diverse screening libraries. Herein we report a newly developed, zirconium tetrakis(dodecyl sulfate) [Zr(DS)4] catalyzed ring-opening of on-DNA epoxides in water with amines, including anilines. Subsequent cyclization of the resulting on-DNA β-amino alcohols leads to a variety of biologically interesting, non-aromatic heterocycles. Using these conditions, a library of 137 million on-DNA β-amino alcohols and their cyclization products was assembled.
      PubDate: 2016-11-30T21:21:43.160988-05:
      DOI: 10.1002/cbic.201600563
       
  • SHIP2: Structure, Function and Inhibition

    • Authors: Mark P Thomas; Christophe Erneux, Barry V L Potter
      Abstract: SHIP2 is a phosphatase that acts at the 5-position of phosphatidylinositol 3,4,5-trisphosphate. It is one of several enzymes that catalyse dephosphorylation at the 5-position of phosphoinositides or inositol phosphates. SHIP2 has a confirmed role in opsismodysplasia, a disease of bone development, but also interacts with proteins involved in insulin signalling, cytoskeletal function (thus having an impact on endocytosis, adhesion, proliferation and apoptosis) and immune system function. The structure of three domains (constituting about 38% of the protein) is known. Inhibitors of SHIP2 activity have been designed to interact with the catalytic domain with sub-micromolar IC50 values: these come from a range of structural classes and have been shown to have in vivo effects consistent with SHIP2 inhibition. Much remains unknown about the roles of SHIP2 and possible future directions for research are indicated.
      PubDate: 2016-11-30T10:11:23.814387-05:
      DOI: 10.1002/cbic.201600541
       
  • A Thioacetal Photocage Designed for Dual Release: Application in the
           Quantitation of Therapeutic Release by Synchronous Reporter Decaging

    • Authors: Pamela T. Wong; Shengzhuang Tang, Jayme Cannon, Jhindan Mukherjee, Danielle Isham, Kristina Gam, Michael Payne, Sean A. Yanik, James R. Baker, Seok Ki Choi
      Abstract: Despite the immense potential of existing photocaging technology, its application is limited by the paucity of advanced caging tools. Here, we report on the design of a novel thioacetal ortho-nitrobenzaldehyde (TNB) dual arm photocage that enabled control of the simultaneous release of two payloads linked to a single TNB unit. By using this cage, which was prepared in a single step from commercial 6-nitroverataldehyde, three drug–fluorophore conjugates were synthesized: Taxol-TNB-fluorescein, Taxol-TNB-coumarin, and doxorubicin-TNB-coumarin, and long-wavelength UVA light-triggered release experiments demonstrated that dual payload release occurred with rapid decay kinetics for each conjugate. In cell-based assays performed in vitro, dual release could also be controlled by UV exposure, resulting in increased cellular fluorescence and cytotoxicity with potency equal to that of unmodified drug towards the KB carcinoma cell line. The extent of such dual release was quantifiable by reporter fluorescence measured in situ and was found to correlate with the extent of cytotoxicity. Thus, this novel dual arm cage strategy provides a valuable tool that enables both active control and real-time monitoring of drug activation at the delivery site.Binary photocage: An ortho-nitrobenzaldehyde-derived dual arm photocage was developed for real-time monitoring of the simultaneous release of two payloads linked to a single cage unit. Light-controlled uncaging of the drug–fluorophore conjugate resulted in increased cellular fluorescence, which was found to correlate with cytotoxicity.
      PubDate: 2016-11-30T09:21:30.054955-05:
      DOI: 10.1002/cbic.201600494
       
  • 2016 EMBO Chemical Biology Conference

    • Authors: Satpal Virdee
      Abstract: The full breadth of the field: The 2016 EMBO Chemical Biology Conference, covering topics from tool development to biological applications and from computational drug design to synthetic chemistry, took place in Heidelberg from 31st August to 3rd September.
      PubDate: 2016-11-30T08:40:50.346599-05:
      DOI: 10.1002/cbic.201600597
       
  • Design of S-Allylcysteine in Situ Production and Incorporation Based on a
           Novel Pyrrolysyl-tRNA Synthetase Variant

    • Authors: Matthias P. Exner; Tilmann Kuenzl, Tuyet Mai T. To, Zhaofei Ouyang, Sergej Schwagerus, Michael G. Hoesl, Christian P. R. Hackenberger, Marga C. Lensen, Sven Panke, Nediljko Budisa
      Abstract: The noncanonical amino acid S-allyl cysteine (Sac) is one of the major compounds of garlic extract and exhibits a range of biological activities. It is also a small bioorthogonal alkene tag capable of undergoing controlled chemical modifications, such as photoinduced thiol-ene coupling or Pd-mediated deprotection. Its small size guarantees minimal interference with protein structure and function. Here, we report a simple protocol efficiently to couple in-situ semisynthetic biosynthesis of Sac and its incorporation into proteins in response to amber (UAG) stop codons. We exploited the exceptional malleability of pyrrolysyl-tRNA synthetase (PylRS) and evolved an S-allylcysteinyl-tRNA synthetase (SacRS) capable of specifically accepting the small, polar amino acid instead of its long and bulky aliphatic natural substrate. We succeeded in generating a novel and inexpensive strategy for the incorporation of a functionally versatile amino acid. This will help in the conversion of orthogonal translation from a standard technique in academic research to industrial biotechnology.A biochemical pathway for production of proteins containing S-allylcysteine: In vivo site-specific protein olefination with S-allylcysteine (Sac), produced in situ from allyl thiol, is possible with a novel pyrrolysyl-tRNA synthetase capable of accepting a small substrate. The S-allyl group is suitable as a minimal tag in different bio-conjugations settings.
      PubDate: 2016-11-30T08:40:48.357023-05:
      DOI: 10.1002/cbic.201600537
       
  • Type III Polyketide Synthases: Functional Classification and
           Phylogenomics

    • Authors: Yugo Shimizu; Hiroyuki Ogata, Susumu Goto
      Abstract: Polyketide synthases (PKSs) catalyze the sequential condensation of simple acetate units to produce a large class of natural products, including pharmacologically valuable compounds. PKSs are classified into three types on the basis of their domain structures; type III PKSs have the simplest domain structure, although their products have various structures and functions. The sequence–function relationship is fundamental for predicting enzyme functions, but it has not been well investigated in type III PKSs to date. Consequently, the current methods for predicting type III PKS functions are still immature in comparison with those that target type I/II PKSs. In this review we summarize the current functional and phylogenomic knowledge about type III PKSs and propose a new classification of their enzymatic reactions. We also discuss possible directions for the development of better computational tools for functional prediction of type III PKS homologues.A large class of natural products, including pharmacologically valuable compounds, is produced by polyketide synthases (PKSs). Methods for predicting type III PKS functions are still evolving. We summarize the current functional and phylogenomic knowledge about type III PKSs, propose a new classification of their enzymatic reactions, and discuss possible functional prediction methods for type III PKS homologues.
      PubDate: 2016-11-30T08:40:45.876186-05:
      DOI: 10.1002/cbic.201600522
       
  • DNA Interstrand Crosslinks by H-pin Polyamide (S)-seco-CBI Conjugates

    • Authors: Chuanxin Guo; Sefan Asamitsu, Gengo Kashiwazaki, Shinsuke Sato, Toshikazu Bando, Hiroshi Sugiyama
      Abstract: Although DNA interstrand crosslinking (ICL) agents are widely used as antitumor drugs, DNA sequence-specific ICL agents are quite rare. In this study, H-pin imidazole-pyrrole polyamide 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-ol (seco-CBI) conjugates that produce sequence-specific DNA ICLs were designed and synthesized. Conjugates with H-pin polyamide and seco-CBI moieties were constructed to recognize a 7 bp DNA sequence, and their reactivity and selectivity in DNA alkylation were evaluated by using high-resolution denaturing gel electrophoresis and sequence-specific plasmid cleavage. One conjugate (6), which contained a chiral (S)-seco-CBI, exhibited greater sequence-specific ICL activity toward the target DNA sequence and was cytotoxic to a cancer cell line. Molecular modeling studies indicated that the greater activity of 6 resulted from the relative orientation of the cyclopropane group in the (S)-CBI unit.Conjugates and crosslinks: H-pin imidazole-pyrrole polyamide seco-CBI conjugates that can be used to form sequence-specific DNA interstrand crosslinks (ICLs) have been synthesized. One conjugate, which contained a chiral (S)-seco-CBI moiety, exhibited the highest sequence-specific ICL activity toward the target DNA sequence and was cytotoxic to a cancer cell line.
      PubDate: 2016-11-30T08:40:34.787193-05:
      DOI: 10.1002/cbic.201600425
       
  • Influence of Label and Charge Density on the Association of the
           Therapeutic Monoclonal Antibodies Trastuzumab and Cetuximab Conjugated to
           Anionic Fluorophores

    • Authors: Jutta Pauli; Marieke Pochstein, Andrea Haase, Joanna Napp, Andreas Luch, Ute Resch-Genger
      Abstract: The design of bright and functional dye–protein conjugates requires hydrophilic and stable fluorophores with high molar absorption coefficients and high fluorescence quantum yields, which must not be prone to dimerization, as well as conservation of protein function and suppression of protein association. Although many synthetic dyes meet these needs, the influence of dye charge on bioconjugate performance is commonly neglected. This encouraged us to assess the spectroscopic properties, antibody functionality, binding behavior, folding, and association of conjugates of the therapeutic antibodies trastuzumab and cetuximab with the red cyanine dyes S0586, S2381, and 6SIDCC (bearing two, three, and six sulfonate groups, respectively). Our results demonstrate a negligible effect of dye labeling on antibody folding, yet a strong influence of label charge and density on antibody isoelectric points and association. Especially 6SIDCC decreased strongly the isoelectric points of both antibodies and their heavy or light chains even at low labeling degrees, thus favoring protein association. Although an increasingly negative dye charge reduces antigen affinity as shown in a competitive immunoassay, all conjugates still bound to cells overexpressing the target of the respective antibody. Obviously, dyes that cause minimum dimerization with a small number of charged groups are best for conjugate brightness, minimum protein association, and strong target binding. This underlines the need to consider dye charge for the rational design of conjugates with optimum performance.Fluorescent labels are commonly conjugated to antibodies; however, the effect of the fluorophore charge on antibody performance is often overlooked. Here we attached cyanine dyes with a varying number of sulfonate groups to the antibodies trastuzumab and cetuximab. Isoelectric points were affected but both antibodies still bound to their cellular targets.
      PubDate: 2016-11-30T04:47:45.736275-05:
      DOI: 10.1002/cbic.201600299
       
  • Peptoid Efficacy against Polymicrobial Biofilms Determined by Using
           Propidium Monoazide-Modified Quantitative PCR

    • Authors: Yu Luo; Hannah L. Bolt, Gabriela A. Eggimann, Danny F. McAuley, Ronan McMullan, Tanya Curran, Mei Zhou, Professor Colin A. B. Jahoda, Steven L. Cobb, Fionnuala T. Lundy
      Abstract: Biofilms containing Candida albicans are responsible for a wide variety of clinical infections. The protective effects of the biofilm matrix, the low metabolic activity of microorganisms within a biofilm and their high mutation rate, significantly enhance the resistance of biofilms to conventional antimicrobial treatments. Peptoids are peptide-mimics that share many features of host defence antimicrobial peptides but have increased resistance to proteases and therefore have better stability in vivo. The activity of a library of peptoids was tested against monospecies and polymicrobial bacterial/fungal biofilms. Selected peptoids showed significant bactericidal and fungicidal activity against the polymicrobial biofilms. This coupled with low cytotoxicity suggests that peptoids could offer a new option for the treatment of clinically relevant polymicrobial infections.Peptoids can mimic host-defence antimicrobial peptides but have increased resistance to proteases. We constructed a library of such peptoids and showed that they reduce the cell numbers of C. albicans, S. aureus and E. coli in polymicrobial biofilms, as quantified by a novel PMA-qPCR assay.
      PubDate: 2016-11-30T04:47:39.558854-05:
      DOI: 10.1002/cbic.201600381
       
  • Effect of Methyl Group on Acyclic Serinol Scaffold for Tethering Dyes on
           the DNA Duplex Stability

    • Authors: Keiji Murayama; Hiroyuki Asanuma
      Abstract: Acyclic serinol derivatives are useful scaffolds for tethering dyes within DNA duplexes. Here we synthesised an inverse l-threoninol (il-threoninol) scaffold and compared its effect on DNA duplex stability to other acyclic artificial nucleic acid scaffolds that are based on d-threoninol, l-threoninol, and serinol. When planar trans-azobenzene was incorporated into the DNA duplex through a single bulge-like motif (the wedge), the il-threoninol scaffold stabilised the duplex most efficiently. When scaffolds were incorporated in complementary positions (dimer motif) or in three adjacent positions (cluster motif), d-threoninol was the most stabilising. CD spectra indicated that the effect of scaffold on the duplex stability was closely related to the winding induced by each scaffold. When trans-azobenzene was photo-isomerised to non-planar cis-azobenzene, il-threoninol destabilised the duplex most strongly, irrespective of the number of artificial residues incorporated. The properties of the il-threoninol scaffold make it a useful tether for dyes or other functionalities.Tie dye: We synthesised an inverse l-threoninol scaffold and compared its effect on DNA duplex stability to other acyclic artificial nucleic acid scaffolds based on d-threoninol and serinol. All three stabilised duplexes by intercalation of planar dye. The results indicate that the il-threoninol scaffold will be useful tether for dyes or other functionalities.
      PubDate: 2016-11-30T04:28:55.976019-05:
      DOI: 10.1002/cbic.201600558
       
  • Transformation of free and dipeptide-bound glycated amino acids by two
           strains of Saccharomyces cerevisiae

    • Authors: Michael Hellwig; Marie Börner, Falco Beer, Karl-Heinz van Pée, Thomas Henle
      Abstract: The yeast Saccharomyces cerevisiae transforms branched-chain and aromatic amino acids to higher alcohols in the Ehrlich pathway. During microbiological culturing and industrial fermentations, this yeast is confronted with amino acids modified by reducing sugars in the Maillard reaction (glycation). In order to get first insights into the physiological "handling" of glycated amino acids by yeasts, individual Maillard reaction products (MRPs; fructosyllysine, carboxymethyllysine, pyrraline, formyline, maltosine, methylglyoxal-derived hydroimidazolone) were administered to two strains of S. cerevisiae in a rich medium. Only formyline was converted to the corresponding α-hydroxy acid to a small extent (10%). Dipeptide-bound pyrraline and maltosine were removed from the medium with concomitant emergence of several metabolites. Pyrraline was mainly converted to the corresponding Ehrlich alcohol (20-60%) and maltosine to the corresponding α-hydroxy acid (40-60%). Five specific metabolites of glycated amino acids were synthesized and characterized. We show for the first time that S. cerevisiae can use glycated amino acids as a nitrogen source and transform them to novel metabolites provided that the substances can be transported across the cell membrane.
      PubDate: 2016-11-30T03:10:32.805837-05:
      DOI: 10.1002/cbic.201600486
       
  • Modern Approaches to Discovering New Hydroxynitrile Lyases for
           Biocatalysis

    • Authors: Santosh Kumar Padhi
      Abstract: Hydroxynitrile lyases (HNLs) have grown in importance from laboratory to industry due to their potential to catalyze stereoselective C−C bond-formation reactions in the synthesis of several chiral intermediates, such as enantiopure α-cyanohydrins, β-nitro alcohols, and their derivatives with multiple functional groups. With these wide applications, the demand for finding new HNLs has increased, and this has led to exploration not only of new HNLs but also of new ways to discover them. An exclusive review article on HNLs by Asano et al. in 2011 described the discovery of HNLs along with their applications. Since then many scientific advancements have been seen in this area. This article aims to highlight the modern HNL discovery approaches, based mainly on 1) genome mining, 2) use of INTMSAlign software, 3) rational design (based on a millipede HNL), 4) evolution of catalytic mechanisms, 5) protein engineering guided by catalytic mechanisms, and 6) screening of plants with cyanogen glycoside (CG) content. This description is followed by future prospects. Overall this review represents the present state and the future potential of HNL discovery approaches, and so might be hoped to be instrumental not only in exploration of new HNLs but also in the invention of methods for potential biotechnological applications.Lyases—enzyme engineering and screening: The diverse approaches used to discover new hydroxynitrile lyases (HNLs) are described. The emphasis is on understanding of modern approaches based on genome mining, use of INTMSAlign software, rational design (based on a millipede HNL), evolution of catalytic mechanisms, etc.
      PubDate: 2016-11-29T09:00:22.464802-05:
      DOI: 10.1002/cbic.201600495
       
  • Structural rearrangement from oligomer to fibril detected with FRET in a
           designed amphiphilic peptide

    • Authors: Heng Chi; Timothy A. Keiderling
      Abstract: ß-sheet conformation is promoted in peptides with amphiphilic design, and stable ß-turn formation is favored with the unnatural amino acid DPro followed by a flexible residue, such as Gly. A 19-residue peptide (B3) was synthesized with alternating hydrophobic and hydrophilic residues connected by symmetrical DPro-Gly and Gly-DPro turns. B3 forms an oligomeric aggregate, rich in ß-sheet conformation, which reversibly transforms into unordered structure on heating as evidenced by its temperature dependent IR spectra. When a dansyl moiety was added to the N-terminus of B3, the resulting peptide (B3D) can convert into a fibrillar structure after higher temperature incubation, which is detectable spectroscopically as well as by TEM. The fibrillization process involved an initial unfolding step monitored by the quenching of dansyl emission that contrasted with subsequent enhanced ThT emission seen on its binding to the fibril. A possible mechanism is proposed in which B3D forms a low-temperature oligomer that is at least partially unfolded due to a heat trigger, and, after this reassembles more slowly as a fibril.
      PubDate: 2016-11-29T08:11:53.051844-05:
      DOI: 10.1002/cbic.201600436
       
  • Bifunctionality of ActIV as a Cyclase-Thioesterase Revealed by In Vitro
           Reconstitution of Actinorhodin Biosynthesis in Streptomyces coelicolor
           A3(2).

    • Authors: Takaaki Taguchi; Takayoshi Awakawa, Yukitaka Nishihara, Michiho Kawamura, Yasuo Ohnishi, Koji Ichinose
      Abstract: Type II polyketide synthase (PKS) iteratively generates a nascent polyketide thioester of acyl carrier protein (ACP), which is structurally modified to produce an ACP-free intermediate towards its final metabolite. However, the timing of ACP off-loading is not well defined due to the lack of an apparent thioesterase (TE) among relevant biosynthetic enzymes. Here, ActIV, which had been assigned as a second ring cyclase (CYC) in actinorhodin (ACT) biosynthesis, was shown to possess a TE activity in vitro with a model substrate, anthraquinone-2-carboxylic acid-N-acetylcysteamine. To further investigate its function, the ACT biosynthetic pathway in Streptomyces coelicolor A3(2) was stepwisely reconstituted in vitro up to (S)-DNPA, and the product of ActIV reaction was characterized as an ACP-free bicyclic intermediate.These findings indicate that ActIV is a bifunctional CYC-TE and provide clear-cut evidence for the release timing of the intermediate from the ACP anchor.
      PubDate: 2016-11-29T08:11:51.881916-05:
      DOI: 10.1002/cbic.201600589
       
  • Active site crowding of P450 3A4 as a strategy to alter its selectivity

    • Authors: Paolo Schiavini; Kin J. Cheong, Nicolas Moitessier, Karine Auclair
      Abstract: Substrate promiscuous enzymes are a promising starting point for the development of versatile biocatalysts. In this study, human cytochrome P450 3A4, known for its ability to metabolize hundreds of drugs, was engineered to alter its regio- and stereoselectivity. Rational mutagenesis was used to specifically introduce steric hindrance in the large active site of P450 3A4 and favour oxidation at a more sterically accessible position on the substrate. Hydroxylation of a synthetic precursor of (R)-lisofylline, a compound under investigation for its anti-inflammatory properties, was chosen as a first proof-of-principle application of our protein engineering strategy. In a second example, increasing active site crowding led to an incremental shift in the selectivity of oxidation from an internal double bond to a terminal phenyl group in a derivative of theobromine. The same correlation between crowding and selectivity was found in a final case focused on the hydroxylation of the steroid sex hormone progesterone.
      PubDate: 2016-11-29T08:11:47.679291-05:
      DOI: 10.1002/cbic.201600546
       
  • Iterative non-proteinogenic residue incorporation yields α/β-peptides
           with a helix-loop-helix tertiary structure and high affinity for VEGF

    • Authors: James W. Checco; Samuel H Gellman
      Abstract: Inhibition of specific protein-protein interactions is attractive for a range of therapeutic applications, but the large and irregularly shaped contact surfaces involved in many such interactions make it challenging to design synthetic antagonists. Here, we describe the development of backbone-modified peptides containing both α- and β-amino acid residues ("α/β-peptides") that target the receptor-binding surface of vascular endothelial growth factor (VEGF). Our approach is based on the Z-domain, which adopts a three-helix bundle tertiary structure. We show how a two-helix "mini-Z-domain" can be modified to contain β and other non-proteinogenic residues while retaining the target-binding epitope using iterative non-natural residue incorporation. The resulting α/β-peptides are less susceptible to proteolysis than is their parent α-peptide, and some of these α/β-peptides match the full-length Z-domain in terms of affinity for receptor-recognition surfaces on the VEGF homodimer.
      PubDate: 2016-11-29T08:11:45.562574-05:
      DOI: 10.1002/cbic.201600545
       
  • Small Molecule-Induced and Cooperative Enzyme Assembly on a 14-3-3
           Scaffold

    • Authors: Anniek den Hamer; Lenne Lemmens, Minke Nijenhuis, Christian Ottmann, Maarten Merkx, Tom de Greef, Luc Brunsveld
      Abstract: Scaffold proteins regulate cell signaling by promoting the proximity of putative interaction partners. While frequently applied in a cellular setting, their fundamental understanding, amongst other in terms of quantitative parameters, has been lagging behind. Here we present a scaffold protein platform based on the native 14-3-3 dimeric protein and under the control of a small-molecule, which allows to be studied in an in vitro setting and mathematically described. Robust small-molecule regulation of caspase-9 activity via induced dimerization on the 14-3-3 scaffold was demonstrated. The individual parameters of this system were precisely determined and used to develop a mathematical model of the scaffolding concept. This model was used to elucidate the strong cooperativity of the 14-3-3 scaffold-mediated enzyme activation. This work provides entry into the long needed quantitative insights in scaffold protein functioning and paves the way for the optimal use of reengineered 14-3-3 proteins as chemical inducible scaffolds in synthetic systems.
      PubDate: 2016-11-29T08:11:41.956784-05:
      DOI: 10.1002/cbic.201600631
       
  • A New Caged-Glutamine Derivative as a Tool To Control the Assembly of
           Glutamine-Containing Amyloidogenic Peptides

    • Authors: Loay Awad; Nino Jejelava, Ritwik Burai, Hilal A. Lashuel
      Abstract: We present the design, synthesis, and characterization of a novel photocaged glutamine derivative (modified on the side chain of glutamine), and describe its use in enhancing peptide stability and solubility. Our results demonstrate that this approach can be used to develop molecular switches to control the folding and β-sheet formation of amyloidogenic peptides.Caged amyloids: A photocaged glutamine derivative has been developed by modifying the side chain of glutamine. It enhances peptide solubility and can be used as a molecular switch to control the folding and formation of β-sheet structures of glutamine-containing amyloidogenic peptides, as demonstrated by TEM.
      PubDate: 2016-11-25T07:45:29.08758-05:0
      DOI: 10.1002/cbic.201600474
       
  • Novel Amino Acid-Based β-Phosphorylated Nitroxides for Probing Acidic pH
           in Biological Systems by EPR Spectroscopy

    • Authors: Sophie Thétiot-Laurent; Gaëlle Gosset, Jean-Louis Clément, Mathieu Cassien, Anne Mercier, Didier Siri, Anouk Gaudel-Siri, Antal Rockenbauer, Marcel CULCASI, Sylvia Pietri
      Abstract: There is increasing attraction of measuring pH in biological studies using nitroxides having pH-dependent electron paramagnetic resonance (EPR) spectra. Aiming to improve the spectral sensitivity, ΔaX, of these probes (i.e. the difference between the EPR hyperfine splittings (hfs) in their protonated and unprotonated forms), we present here a series of novel linear α-carboxy, α'-diethoxyphosphoryl nitroxides constructed on an amino acid core and featuring a (α or α')-C−H bond. In buffers the three main hfs (aN, aH and aP) of their EPR spectra vary reversibly with pH and, when it is inferred from aP or aH titration curves, a 2−4-fold increase in sensitivity is achieved vs reference imidazoline or imidazolidine nitroxides. The crystallized carboxylate 10b (pKa ≈ 3.6), which demonstrated low cytotoxicity and good resistance to bioreduction, was applied to probe the stomach acidity in rats. The results pave the way to a novel generation of highly sensitive EPR pH markers.
      PubDate: 2016-11-25T02:41:58.992645-05:
      DOI: 10.1002/cbic.201600550
       
  • Bivalent display of dicysteine on PNA for homogenous DNA/RNA detection via
           in situ fluorescence labelling

    • Authors: Gemin Fang; Oliver Seitz
      Abstract: Fluorogenic probes that signal the presence of specific DNA or RNA sequences are key enabling tools for molecular disease diagnosis and imaging studies. Usually, at least one fluorophore is attached via covalent bonds to an oligonucleotide probe. However, the additional conjugation step increases costs. Herein we introduce a method that avoids the requirement for the preparation of fluorescence labeled oligonucleotides and provides the opportunity to alter the fluorogenic reporter dye without resynthesis. The method is based on adjacent hybridization of two dicysteine-containing peptide nucleic acid probes to form a bipartite tetracysteine motif which binds profluorescent bisarsenical dyes such as FIAsH, ReAsH or CrAsH. Binding is accompanied by strong increases of fluorescence emission (with response factors up to 80-fold and high brightness up to 50 mL·mol−1·cm−1). The detection system provides subnanomolar limits of detection and allows discrimination of single nucleotide variations by more than 20-fold changes of fluorescence intensity. To demonstrate the usefulness, the FIAsH-based read-out of the bivalent CysCys-PNA display was interfaced with a rolling circle amplification (RCA) assay used to detect disease-associated microRNA let-7a.
      PubDate: 2016-11-24T06:41:09.674297-05:
      DOI: 10.1002/cbic.201600623
       
  • TET1-Mediated Oxidation of 5-Formylcytosine (5fC) to 5-Carboxycytosine
           (5caC) in RNA

    • Authors: Maria Basanta-Sanchez; Rui Wang, Zhenzhen Liu, Xiaohan Ye, Minyong Li, Xiaodong Shi, Paul F. Agris, Yubin Zhou, Yun Huang, Jia Sheng
      Abstract: It was recently revealed that 5-methylcytosine (5mC) in mRNA, similar to its behavior in DNA, can be oxidized to produce 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC), implying the potential regulatory roles of this post-transcriptional RNA modification. In this study, we demonstrate the in vitro oxidation of 5fC to 5-carboxycytidine (5caC) by the catalytic domain of mammalian ten-eleven translocation enzyme (TET1) in different RNA contexts. We observed that this oxidation process has very low sequence dependence and can take place in single-stranded, double-stranded, or hairpin forms of RNA sequences, although the overall conversion yields are low.Not depending on the context: The mammalian ten–eleven translocation enzyme TET1 can oxidize 5-formylcytidine (5frC) to 5-carboxycytidine (5carC) in different RNA contexts. This oxidation process can take place in single-stranded, double-stranded, or hybrid forms of RNA sequences.
      PubDate: 2016-11-22T08:50:25.426016-05:
      DOI: 10.1002/cbic.201600328
       
  • Coiled-coil mediated activation of oligo-arginine cell-penetrating
           peptides

    • Authors: Saskia Bode; Ilmar Kruis, Hans Adams, Wilbert Boelens, Ger Pruijn, Jan Van Hest, Dennis Lowik
      Abstract: A supramolecular approach was undertaken to create functionally activatable cell-penetrating peptides. Two tetra-arginines were assembled into an active cell-penetrating peptide by hetero-dimerizing leucine zippers. Three different leucine zipper pairs were evaluated of which the activation was found to be dependent on the association constant of the coiled-coil peptides. The weaker binding peptides required an additional disulfide linkage to educe cell-penetrating capacity, whereas for the most stable coiled-coil no additional stabilization was needed. The latter zipper pair was used to show that the induced formation of the coiled-coils allows to control the uptake of an oligo-arginine CPP-conjugated cargo protein.
      PubDate: 2016-11-21T12:30:20.366807-05:
      DOI: 10.1002/cbic.201600614
       
  • Structure activity relationships of benzenesulfonamide-based inhibitors
           towards carbonic anhydrase isoform specificity

    • Authors: Avni Bhatt; Brian P. Mahon, Vinicius Wilian D. Cruzeiro, Benedetta Cornelio, Marie Laronze-Cochard, Mariangela Ceruso, Janos Sapi, Graham A. Rance, Andrei N. Khlobystov, Antonella Fontana, Adrian Roitberg, Claudiu T. Supuran, Robert Mc Kenna
      Abstract: Carbonic anhydrases (CAs) are implicated in a wide range of diseases, including the upregulation of isoforms CA IX and XII in many aggressive cancers. However, effective inhibition of disease-implicated CAs should minimally affect the ubiquitously expressed isoforms including CA I and II to improve directed distribution of the inhibitors to the cancer-associated isoforms and reduce side effects. Four benzenesulfonamide-based inhibitors were synthesized using the "tail approach" and display nM affinities for several CA isoforms. The crystal structures of the inhibitors bound to a CA IX-mimic and CA II are presented. Further in-silico modeling was performed with the inhibitors docked into CA I and XII to identify residues that contribute to or hinder their binding interactions. These structural studies demonstrate that active site residues lining the hydrophobic pocket, especially positions 92 and 131, dictate the positional binding and affinity of inhibitors and the tail groups modulate CA isoform specificity. Geometry optimizations were performed on each ligand in the crystal structures and showed the energetic penalties of the inhibitors' conformations were negligible compared to the gain of active site interactions. These studies further our understanding of obtaining isoform specificity when designing small molecule CA inhibitors.
      PubDate: 2016-11-18T05:15:33.100082-05:
      DOI: 10.1002/cbic.201600513
       
  • Protein surface mimetics: understanding how ruthenium tris(bipyridines)
           interact with proteins

    • Authors: Sarah H Hewitt; Maria H Filby, Ed Hayes, Lars T Kuhn, Arnout P. Kalverda, Michael E Webb, Andrew John Wilson
      Abstract: : Protein surface mimetics achieve high affinity binding by exploiting a scaffold to project binding groups over a large area of solvent exposed protein surface to make multiple co-operative non-covalent interactions. Such recognition is a pre-requisite for competitive/ orthosteric inhibition of protein-protein interactions (PPIs). This paper describes biophysical and structural studies on ruthenium(II) tris(bipyridine) surface mimetics that recognize cytochrome (cyt) c and inhibit the cyt c/ cyt c peroxidase (CCP) PPI. Binding is electrostatically driven, with enhanced affinity achieved through enthalpic contributions thought to arise from the ability of the surface mimetics to make a greater number of non-covalent interactions with surface exposed basic residues on cyt c in comparison to CCP. High field natural abundance 1H-15N HSQC NMR experiments are consistent with surface mimetics binding to cyt c in similar manner to CCP. This provides a framework for understanding recognition of proteins by supramolecular receptors and informing the design of ligands superior to the protein partners upon which they are inspired.
      PubDate: 2016-11-17T11:35:23.813484-05:
      DOI: 10.1002/cbic.201600552
       
  • Copper is a cofactor of the formylglycine generating enzyme

    • Authors: Matthias Knop; Thanh Quy Quy Dang, Gunnar Jeschke, Florian P. Seebeck
      Abstract: The formylglycine generating enzyme (FGE) is an O2 utilizing oxidase that converts specific cysteine residues of client proteins to formylglycine. In this report we show that Cu(I) is an integral cofactor of this enzyme and binds with high affinity (KD = of 10-17 M) to a pair of active site cysteines. These findings establish FGE as a novel type of copper enzyme.
      PubDate: 2016-11-16T02:34:50.057703-05:
      DOI: 10.1002/cbic.201600359
       
  • Photoreduction of Shewanella oneidensis Extracellular Cytochromes by
           Organic Chromophores and Dye-Sensitized TiO2

    • Authors: Emma V. Ainsworth; Colin W. J. Lockwood, Gaye F. White, Ee Taek Hwang, Tsubasa Sakai, Manuela A. Gross, David J. Richardson, Thomas A. Clarke, Lars J. C. Jeuken, Erwin Reisner, Julea N. Butt
      Abstract: The inside back cover picture shows the photoreduction of electrically conductive proteins spanning a bacterial outer membrane. The authors show that abiotic and cell-secreted photosensitizers drive the photocatalytic reduction of extracellular cytochromes. Their studies suggest how photoenergized electrons may be delivered to the interior of non-photosynthetic micro-organisms for the synthesis of solar chemicals. More information can be found in the full paper by L. J. C. Jeuken, E. Reisner, J. N. Butt et al. (
      DOI : 10.1002/cbic.201600339).
      PubDate: 2016-11-15T08:13:06.244272-05:
       
  • A Tandem Green–Red Heterodimeric Fluorescent Protein with High FRET
           Efficiency

    • Authors: Matthew D. Wiens; Yi Shen, Xi Li, M. Alaraby Salem, Nick Smisdom, Wei Zhang, Alex Brown, Robert E. Campbell
      Abstract: The tetrameric red fluorescent protein from Discosoma sp. coral (DsRed) has previously been engineered to produce dimeric and monomeric fluorescent variants with excitation and emission profiles that span the visible spectrum. The brightest of the effectively monomeric DsRed variants is tdTomato—a tandem fusion of a dimeric DsRed variant. Here we describe the engineering of brighter red (RRvT), green (GGvT), and green–red heterodimeric (GRvT) tdTomato variants. GRvT exhibited 99 % intramolecular FRET efficiency, resulting in long Stokes shift red fluorescence. These new variants could prove useful for multicolor live-cell imaging applications.Fluorescent fruit: Three tandem dimer fluorescent proteins, designated “vine Tomatoes”, were engineered and photophysically characterized. These proteins can serve as model systems for studying FRET between chromophores with fixed orientations and could prove to be useful for live-cell fluorescence imaging.
      PubDate: 2016-11-15T05:56:20.46786-05:0
      DOI: 10.1002/cbic.201600492
       
  • Tetrazine Responsive Self-Immolative Linkers

    • Authors: Kevin Neumann; Sarthak Jain, Alessia Gambardella, Sarah Walker, Elsa Valero, Annamaria Lilienkampf, Mark Bradley
      Abstract: Molecules that undergo activation or modulation following the addition of benign external small molecule chemical stimuli have numerous of applications. Here we report the highly efficient "decaging" of a variety of moieties by activation of a "self-immolative" linker by application of a water soluble and stable tetrazine, including the controlled delivery of doxorubicin in a cellular context.
      PubDate: 2016-11-14T07:29:48.491152-05:
      DOI: 10.1002/cbic.201600560
       
  • Improving the Binding Affinity of in-Vitro-Evolved Cyclic Peptides by
           Inserting Atoms into the Macrocycle Backbone

    • Authors: Jonas Wilbs; Simon J. Middendorp, Christian Heinis
      Abstract: Cyclic peptides binding to targets of interest can be generated efficiently with powerful in vitro display techniques, such as phage display or mRNA display. The cyclic peptide libraries screened with these methods are generated by altering in a combinatorial fashion the amino acid sequence of the peptides, the number of amino acids in the macrocycle rings, and the cyclization chemistry. A structural element that cannot easily be varied in the cyclic peptides is the backbone, which is built from amino acids, each of which contributes three atoms to the macrocyclic ring structure. Here, we proposed to improve the affinity of a phage-selected bicyclic peptide inhibitor of coagulation factor XII (FXII) by screening variants with one or two carbon atoms inserted into different positions of the backbone, and thus tapping into a structural space that was not sampled by phage display. Two mutants showed 4.7- and 2.5-fold improved Ki values. The better one blocked FXII with a Ki of 1.5±0.1 nm and inhibited activation of the intrinsic coagulation pathway (EC2x 1.7 μm). The strategy of ring size variation by one or several atoms should be generally applicable for the affinity maturation of in-vitro-evolved cyclic peptides.The binding affinity of a bicyclic peptide inhibitor of coagulation factor XII, previously developed by phage display, was improved by screening variants with one or two carbon atoms inserted into different positions of the backbone.
      PubDate: 2016-11-11T07:15:50.651146-05:
      DOI: 10.1002/cbic.201600336
       
  • Identification of Small-Molecule PHD2 Zinc Finger Inhibitors that Activate
           Hypoxia Inducible Factor

    • Authors: Patrick R. Arsenault; Daisheng Song, Marian Bergkamp, Andrew M. Ravaschiere, Bradleigh E. Navalsky, Paul M. Lieberman, Frank S. Lee
      Abstract: The prolyl hydroxylase domain (PHD) protein:hypoxia inducible factor (HIF) pathway is the main pathway by which changes in oxygen concentration are transduced to changes in gene expression. In mammals, there are three PHD paralogues, and PHD2 has emerged as a particularly critical one for regulating HIF target genes such as erythropoietin (EPO), which controls red cell mass and hematocrit. PHD2 is distinctive among the three PHDs in that it contains an N-terminal MYND-type zinc finger. We have proposed that this zinc finger binds a Pro-Xaa-Leu-Glu (PXLE) motif found in proteins of the HSP90 pathway to facilitate HIF-α hydroxylation. Targeting this motif could provide a means of specifically inhibiting this PHD isoform. Here, we screened a library of chemical compounds for their capacity to inhibit the zinc finger of PHD2. We identified compounds that, in vitro, can inhibit PHD2 binding to a PXLE-containing peptide and induce activation of HIF. Injection of one of these compounds into mice induces an increase in hematocrit. This study offers proof of principle that inhibition of the zinc finger of PHD2 can provide a means of selectively targeting PHD2 to activate the HIF pathway.Give me a HIFive! The zinc finger of prolyl hydroxylase domain 2 (PHD2) plays a critical role in the oxygen-dependent control of hypoxia inducible factors (HIFs). Using a novel high-throughput screen and cell and animal models, we identified small-molecule inhibitors of this domain and showed that one led to increased red cell mass, a hallmark of HIF activation.
      PubDate: 2016-11-11T07:10:24.039593-05:
      DOI: 10.1002/cbic.201600493
       
  • Switch in Cofactor Specificity of a Baeyer–Villiger Monooxygenase

    • Authors: Andy Beier; Sven Bordewick, Maika Genz, Sandy Schmidt, Tom van den Bergh, Christin Peters, Henk-Jan Joosten, Uwe T. Bornscheuer
      Abstract: Baeyer–Villiger monooxygenases (BVMOs) catalyze the oxidation of ketones to esters or lactones by using molecular oxygen and a cofactor. Type I BVMOs display a strong preference for NADPH. However, for industrial purposes NADH is the preferred cofactor, as it is ten times cheaper and more stable. Thus, we created a variant of the cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 (CHMOAcineto); this used NADH 4200-fold better than NADPH. By combining structure analysis, sequence alignment, and literature data, 21 residues in proximity of the cofactor were identified and targeted for mutagenesis. Two combinatorial variants bearing three or four mutations showed higher conversions of cyclohexanone with NADH (79 %) compared to NADPH (58 %) as well as specificity. The structural reasons for this switch in cofactor specificity of a type I BVMO are especially a hydrogen-bond network coordinating the two hydroxy groups of NADH through direct interactions and bridging water molecules.Switch the cofactor preference! Variants of the cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 were engineered to accept NADH significantly better than NADPH. By using a computational design, 21 positions were subjected to mutagenesis thereby resulting in a quadruple mutant with a 4200-fold improvement in cofactor preference for NADH.
      PubDate: 2016-11-09T15:38:32.491507-05:
      DOI: 10.1002/cbic.201600484
       
  • Photoreduction of Shewanella oneidensis Extracellular Cytochromes by
           Organic Chromophores and Dye-Sensitized TiO2

    • Authors: Emma V. Ainsworth; Colin W. J. Lockwood, Gaye F. White, Ee Taek Hwang, Tsubasa Sakai, Manuela A. Gross, David J. Richardson, Thomas A. Clarke, Lars J. C. Jeuken, Erwin Reisner, Julea N. Butt
      Abstract: The transfer of photoenergized electrons from extracellular photosensitizers across a bacterial cell envelope to drive intracellular chemical transformations represents an attractive way to harness nature's catalytic machinery for solar-assisted chemical synthesis. In Shewanella oneidensis MR-1 (MR-1), trans-outer-membrane electron transfer is performed by the extracellular cytochromes MtrC and OmcA acting together with the outer-membrane-spanning porin⋅cytochrome complex (MtrAB). Here we demonstrate photoreduction of solutions of MtrC, OmcA, and the MtrCAB complex by soluble photosensitizers: namely, eosin Y, fluorescein, proflavine, flavin, and adenine dinucleotide, as well as by riboflavin and flavin mononucleotide, two compounds secreted by MR-1. We show photoreduction of MtrC and OmcA adsorbed on RuII-dye-sensitized TiO2 nanoparticles and that these protein-coated particles perform photocatalytic reduction of solutions of MtrC, OmcA, and MtrCAB. These findings provide a framework for informed development of strategies for using the outer-membrane-associated cytochromes of MR-1 for solar-driven microbial synthesis in natural and engineered bacteria.Pump priming: Outer-membrane cytochromes provide conduits for electron exchange between bacteria and their environment. Photoreduction of these cytochromes has been achieved. This research paves the way to light-driven microbial synthesis by use of extracellular photosensitizers to harness nature's versatile catalytic machinery.
      PubDate: 2016-11-08T04:16:43.11872-05:0
      DOI: 10.1002/cbic.201600339
       
  • Guide Strand 3′-End Modifications Regulate siRNA Specificity

    • Authors: Rachel A. P. Valenzuela; Kazumitsu Onizuka, Alexi A. Ball-Jones, Tiannan Hu, Scott R. Suter, Peter A. Beal
      Abstract: Short interfering RNA (siRNA)-triggered gene knockdown through the RNA interference (RNAi) pathway is widely used to study gene function, and siRNA-based therapeutics are in development. However, as the guide strand of an siRNA can function like a natural microRNA (miRNA), siRNAs often repress hundreds of off-target transcripts with complementarity only to the seed region (nucleotides 2–8) of the guide strand. Here, we describe novel guide strand 3′-end modifications derived from 1-ethynylribose (1-ER) and copper-catalyzed azide–alkyne cycloaddition reactions and evaluate their impact on target versus miRNA-like off-target knockdown. Surprisingly, when positioned at the guide strand 3′-end, the parent 1-ER modification substantially reduced off-target knockdown while having no measurable effect on on-target knockdown potency. In addition, these modifications were shown to modulate siRNA affinity for the hAgo2 PAZ domain. However, the change in PAZ domain binding affinity was not sufficient to predict the modification's effect on miRNA-like off targeting.The End: 1-Ethynyl ribose (1-ER) was incorporated into a short interfering RNA (siRNA) guide strand at the 3′-end and further modified by azide–alkyne cycloaddition reactions. 1-ER at the 3′-terminal position significantly reduced miRNA off targeting while maintaining on-target knockdown potency.
      PubDate: 2016-11-07T08:21:26.960498-05:
      DOI: 10.1002/cbic.201600453
       
  • Isomeric Detergent Comparison for Membrane Protein Stability: Importance
           of Inter-Alkyl-Chain Distance and Alkyl Chain Length

    • Authors: Kyung Ho Cho; Parameswaran Hariharan, Jonas S. Mortensen, Yang Du, Anne K. Nielsen, Bernadette Byrne, Brian K. Kobilka, Claus J. Loland, Lan Guan, Pil Seok Chae
      Abstract: Membrane proteins encapsulated by detergent micelles are widely used for structural study. Because of their amphipathic property, detergents have the ability to maintain protein solubility and stability in an aqueous medium. However, conventional detergents have serious limitations in their scope and utility, particularly for eukaryotic membrane proteins and membrane protein complexes. Thus, a number of new agents have been devised; some have made significant contributions to membrane protein structural studies. However, few detergent design principles are available. In this study, we prepared meta and ortho isomers of the previously reported para-substituted xylene-linked maltoside amphiphiles (XMAs), along with alkyl chain-length variation. The isomeric XMAs were assessed with three membrane proteins, and the meta isomer with a C12 alkyl chain was most effective at maintaining solubility/stability of the membrane proteins. We propose that interplay between the hydrophile–lipophile balance (HLB) and alkyl chain length is of central importance for high detergent efficacy. In addition, differences in inter-alkyl-chain distance between the isomers influence the ability of the detergents to stabilise membrane proteins.Isomeric detergent comparison enabled us to discover new detergent design principles, by synthesising and testing meta and ortho isomers of our previous para-substituted xylene-linked maltoside amphiphiles. Our results will facilitate the design of new detergents with enhanced membrane-protein stabilisation efficacy.
      PubDate: 2016-11-07T04:33:03.316807-05:
      DOI: 10.1002/cbic.201600429
       
  • Synthesis and Electron Paramagnetic Resonance Studies of
           Oligodeoxynucleotides Containing
           2-N-tert-Butylaminoxyl-2′-deoxyadenosines

    • Authors: Manami Kurita; Yoshitaka Higuchi, JW Mirc, Shintaro Matsumoto, Kazuteru Usui, Hiroshi Suemune, Mariko Aso
      Abstract: Oligodeoxynucleotides (ODNs) containing 2-N-tert-butylaminoxyl-2′-deoxyadenosine (A*) residues were synthesized to allow accurate monitoring of adenine motion by EPR spectroscopy through the agency of direct linkage of the acyclic aminoxyl group to the nucleobase, and EPR studies of the ODNs in single- and double-stranded forms were performed. Upon duplex formation, peak broadening and decreases in peak height were observed in EPR spectra, and the synthesized ODNs were shown to be excellent monitors of hybridization. Comparison of peak height and the h1/h0 signal ratio provided information on the relative mobility of A* in duplexes with different stability. A second set of ODNs each containing two A* residues at different intervals and four dA residues were also synthesized. For these ODNs, correlations were observed between the EPR spectral shapes of the duplexes and the number of dA residues between A* residues, thus demonstrating the potential of A* residues in monitoring of the structures of nucleic acids.Spin-labeled and doubly spin-labeled ODNs containing 2-N-tert-butylaminoxyl-2′-deoxyadenosine (A*), which contains an acyclic amoxyl radical directly connected to the adenine base, provided information on the relative mobility of A* (for single-labeled) in ODNs and on the distances between A* residues (for doubly labeled) in duplexes by comparing peaks.
      PubDate: 2016-11-03T09:23:19.247044-05:
      DOI: 10.1002/cbic.201600465
       
  • Substitution of Two Active-Site Residues Alters C9-Hydroxylation in a
           Class II Diterpene Synthase

    • Authors: Sibongile Mafu; Emil Fischer, J. Bennett Addison, Isabel Riberio Barbosana, Philipp Zerbe
      Abstract: Diterpenes form a vast and diverse class of natural products of both ecological and economic importance. Class II diterpene synthase (diTPS) enzymes control the committed biosynthetic reactions underlying diterpene chemical diversity. Homology modelling with site-directed mutagenesis identified two active-site residues in the horehound (Marrubium vulgare) class II diTPS peregrinol diphosphate synthase (MvCPS1); residue substitutions abolished the unique MvCPS1-catalysed water-capture reaction at C9 and redirected enzyme activity toward formation of an alternative product, halima-5(10),13-dienyl diphosphate. These findings contributed new insight into the steric interactions that govern diTPS-catalysed regiospecific oxygenation reactions and highlight the feasibility of diTPS engineering to provide a broader spectrum of bioactive diterpene natural products.Diterpene synthase catalytic plasticity: Structure-function analysis of the Marrubium vulgare class II diterpene synthase peregrinol diphosphate synthase (MvCPS1) identified two active-site residues that contribute to the enzyme's product specificity by redirecting catalysis from peregrinol diphosphate to the formation of an alternate diterpene scaffold, halima-5(10),13-dienyl diphosphate.
      PubDate: 2016-10-31T08:26:21.527838-05:
      DOI: 10.1002/cbic.201600419
       
  • Structural Basis for Phospholyase Activity of a Class III
           Transaminase Homologue

    • Authors: Anibal Cuetos; Fabian Steffen-Munsberg, Juan Mangas Sanchez, Amina Frese, Uwe T. Bornscheuer, Matthias Höhne, Gideon Grogan
      Abstract: Pyridoxal-phosphate (PLP)-dependent enzymes catalyse a remarkable diversity of chemical reactions in nature. A1RDF1 from Arthrobacter aurescens TC1 is a fold type I, PLP-dependent enzyme in the class III transaminase (TA) subgroup. Despite sharing 28 % sequence identity with its closest structural homologues, including β-alanine:pyruvate and γ-aminobutyrate:α-ketoglutarate TAs, A1RDF1 displayed no TA activity. Activity screening revealed that the enzyme possesses phospholyase (E.C. 4.2.3.2) activity towards O-phosphoethanolamine (PEtN), an activity described previously for vertebrate enzymes such as human AGXT2L1, enzymes for which no structure has yet been reported. In order to shed light on the distinctive features of PLP-dependent phospholyases, structures of A1RDF1 in complex with PLP (internal aldimine) and PLP⋅PEtN (external aldimine) were determined, revealing the basis of substrate binding and the structural factors that distinguish the enzyme from class III homologues that display TA activity.Enzyme evolution and catalytic diversity: The pyridoxal-phosphate-dependent class III transaminase homologue A1RDF1 does not catalyse transamination reactions, but rather the elimination of phosphate from O-phosphoethanolamine. We describe the structural determinants of reaction specificity, with consequences for studies on the human phospholyase AGXT2L1, an important enzyme in phospholipid metabolism.
      PubDate: 2016-10-31T08:22:00.339027-05:
      DOI: 10.1002/cbic.201600482
       
  • Engineering Protein Self-Assembly: A New Approach for the Design of
           Octahedral Cages

    • Authors: Fabien B. L. Cougnon
      Abstract: A new symmetry-based approach allowed the self-assembly of an octahedral protein nanostructure. C3 trimeric and C4 tetrameric oligomerization domains can be combined in an engineered protein to direct assembly into a desired object. This work might provide the basis for a more general and flexible strategy to control protein self-assembly.
      PubDate: 2016-10-28T08:25:41.416576-05:
      DOI: 10.1002/cbic.201600526
       
  • Cover Picture: Exploring the Potent Inhibition of CTP Synthase by
           Gemcitabine-5′-Triphosphate (ChemBioChem 23/2016)

    • Authors: Gregory D. McCluskey; Samy Mohamady, Scott D. Taylor, Stephen L. Bearne
      Pages: 2206 - 2206
      Abstract: The cover picture shows the potent inhibition of the enzyme CTP synthase (yellow) by gemcitabine triphosphate, a metabolite of the chemotherapeutic drug gemcitabine. Gemcitabine is catabolized into dF-dUTP in vivo; however, CTP synthase catalyzes the recycling of dF-dUTP to gemcitabine triphosphate in a glutamine-dependent manner in vitro. Inhibition by gemcitabine triphosphate is ablated by mutation of a single residue in the active site. More information can be found in the full paper by S. L. Bearne et al. on page 2240 in Issue 23, 2016 (
      DOI : 10.1002/cbic.201600405).
      PubDate: 2016-11-22T04:25:24.911131-05:
       
  • Inside Cover: Site-Specific Immobilization of the Peptidoglycan Synthase
           PBP1B on a Surface Plasmon Resonance Chip Surface (ChemBioChem 23/2016)

    • Authors: Inge L. van't Veer; Nadia O. L. Leloup, Alexander J. F. Egan, Bert J. C. Janssen, Nathaniel I. Martin, Waldemar Vollmer, Eefjan Breukink
      Pages: 2207 - 2207
      Abstract: The inside cover picture shows a schematic representation of the creation of a protein with a site specifically incorporated unnatural amino acid containing an azide functionality by using nonsense suppression mutagenesis (upper left). This creates the possibility to immobilize the protein on a cyclooctyne-functionalized surface plasmon resonance (SPR) chip surface in a homogeneous way with respect to its orientation (lower). This is used to measure protein–protein interactions by SPR (upper right). More information can be found in the full paper by E. Breukink et al. on page 2250 in Issue 23, 2016 (
      DOI : 10.1002/cbic.201600461).
      PubDate: 2016-11-23T07:40:45.85307-05:0
       
  • Examination of Potential Exceptions to the F and S Biosynthetic
           Classification of Fused-Ring Aromatic Polyketides

    • Authors: Robert Thomas
      Pages: 2208 - 2215
      Abstract: The reported acetate-derived labelling of the fungal naphthalene γ-pyrone fonsecin, two streptomycete dodecaketide αpyrones TW93f and TW93g, and the streptomycete phenanthraquinones piloquinone, murayaquinone and haloquinone appear to be exceptions to the generalisation that fungi and streptomycetes produce fused-ring aromatic polyketides by different modes of cyclisation. A review of their 1) originally assigned formulae, 2) [13C2]acetate-derived labelling patterns, and 3) modes of cyclisation leads to the recognition of feasible alternative chemical structures or biosynthetic pathways, which are in accord with the originally proposed classification system.The biosynthesis of fused-ring aromatic polyketides is generally classified as either mode F or mode S, depending on the producing organism (fungal or bacterial, respectively). The mechanisms of ring closure are reviewed along with potential exceptions to the rule.
      PubDate: 2016-10-18T07:35:22.228382-05:
      DOI: 10.1002/cbic.201600315
       
  • Light-Activated Triazabutadienes for the Modification of a Viral Surface

    • Authors: Stephanie M. Jensen; Flora W. Kimani, John C. Jewett
      Pages: 2216 - 2219
      Abstract: Chemical crosslinking is a versatile tool for the examination of biochemical interactions, in particular host–pathogen interactions. We report the critical first step toward the goal of probing these interactions by the synthesis and use of a new heterobifunctional crosslinker containing a triazabutadiene scaffold. The triazabutadiene is stable to protein conjugation and liberates a reactive aryl diazonium species upon irradiation with 350 nm light. We highlight the use of this technology by modifying the surface of several proteins, including the dengue virus envelope protein.Going viral with protein modifications: Aryl diazonium ions are masked in the form of triazabutadienes and appended onto proteins. The aryl diazonium ions are released by light and then react with electron-rich aromatic moieties. This is the first step toward a new strategy for catch-and-release pull-down studies.
      PubDate: 2016-10-21T06:00:53.769315-05:
      DOI: 10.1002/cbic.201600508
       
  • Protecting Triazabutadienes To Afford Acid Resistance

    • Authors: Lindsay E. Guzman; Flora W. Kimani, John C. Jewett
      Pages: 2220 - 2222
      Abstract: Recent work on triazabutadienes has shown that they have the ability to release aryl diazonium ions under exceptionally mild acidic conditions. There are instances that require that this release be prevented or minimized. Accordingly, a base-labile protection strategy for the triazabutadiene is presented. It affords enhanced synthetic and practical utility of the triazabutadiene. The effects of steric and electronic factors in the rate of removal are discussed, and the triazabutadiene protection is shown to be compatible with the traditional acid-labile protection strategy used in solid phase peptide synthesis.Behind the mask: Triazabutadienes can be used as masked tyrosine-selective aryl diazonium ions. We report a base-labile protecting group for triazabutadienes to prevent the acid-dependent release of aryl diazonium ions. The pH-dependent deprotection rate constants are reported, thus enabling widespread use of triazabutadienes in chemical biology.
      PubDate: 2016-10-21T06:01:08.509171-05:
      DOI: 10.1002/cbic.201600517
       
  • Efficient Transfection of siRNA by Peptide Dendrimer–Lipid
           Conjugates

    • Authors: Albert Kwok; Gabriela A. Eggimann, Marc Heitz, Jean-Louis Reymond, Florian Hollfelder, Tamis Darbre
      Pages: 2223 - 2229
      Abstract: Efficient delivery of small interfering RNA (siRNA) into cells is the basis of target-gene-specific silencing and, ultimately, gene therapy. However, current transfection reagents are relatively inefficient, and very few studies provide the sort of systematic understanding based on structure–activity relationships that would provide rationales for their improvement. This work established peptide dendrimers (administered with cationic lipids) as siRNA transfection reagents and recorded structure–activity relationships that highlighted the importance of positive charge distribution in the two outer layers and a hydrophobic core as key features for efficient performance. These dendrimer-based transfection reagents work as well as highly optimised commercial reagents, yet show less toxicity and fewer off-target effects. Additionally, the degrees of freedom in the synthetic procedure will allow the placing of decisive recognition features to enhance and fine-tune transfection and cell specificity in the future.Affection for transfection: Peptide dendrimers, administered with cationic lipids, were found to transfer siRNA into living cells and achieve gene knockdown with efficiency comparable to that of commercial reagents. These new transfection agents also had less toxicity and fewer off-target effects than their commercial counterparts and were compatible with structure–function relationship studies.
      PubDate: 2016-11-09T15:38:23.880785-05:
      DOI: 10.1002/cbic.201600485
       
  • DNA-Templated Synthesis of Perylenediimide Stacks Utilizing Abasic Sites
           as Binding Pockets and Reactive Sites

    • Authors: Tadao Takada; Misa Ido, Akane Ashida, Mitsunobu Nakamura, Kazushige Yamana
      Pages: 2230 - 2233
      Abstract: DNA is considered to be a promising biomolecule as a template and scaffold for arranging and organizing functional molecules on the nanoscale. The construction and evaluation of DNAs containing multiple functional molecules that are useful for optoelectronic devices and sensors has been studied. In this paper we report the efficient incorporation of perylenediimide (PDI) units into DNA by using abasic sites both as binding sites and as reactive sites and the construction of PDI stacks within the DNA structure, accomplished through the preorganization of the PDI units in the hydrophobic pocket within the DNA. Our approach could become a valuable method for construction of DNA/chromophore hybrid structures potentially useful for the design of DNA-based devices and biosensors.DNA/chromophore hybrid structures: Efficient reactions between amino- perylenediimide (aPDI) dyes and DNA duplexes containing abasic nucleosides are described. The abasic sites created in the DNA serve as binding pockets and reactive sites, thus affording a useful method for the construction of PDI stacks within the DNA.
      PubDate: 2016-10-14T06:36:21.63098-05:0
      DOI: 10.1002/cbic.201600454
       
  • Reassigning Sense Codon AGA to Encode Noncanonical Amino Acids in
           Escherichia coli

    • Authors: Yiyan Wang; Meng-Lin Tsao
      Pages: 2234 - 2239
      Abstract: A new method has been developed to reassign the rare codon AGA in Escherichia coli by engineering an orthogonal tRNA/aminoacyl–tRNA synthetase pair derived from Methanocaldococcus jannaschii. The tRNA mutant was introduced with a UCU anticodon, and the synthetase was evolved to correctly recognize the modified tRNA anticodon loop and to selectively charge a target noncanonical amino acid (NAA) onto the tRNA. In order to maximize the efficiency of AGA codon reassignment, while avoiding the lethal effects caused by global codon reassignment in cellular proteins, an inducible promoter (araBAD) was utilized to provide temporal controls for overexpression of the aminoacyl–tRNA synthetase and switch on codon reassignment. Using this system, we were able to efficiently incorporate p-acetylphenylalanine, O-methyl-tyrosine, and p-iodophenylalanine into proteins in response to AGA codons. Also, we found that E. coli strain GM10 was optimal in achieving the highest AGA reassignment rates. The successful reassignment of AGA codons reported here provides a new avenue to further expand the genetic code.Teaching an old codon new tricks: AGA, a codon originally coding for arginine, was altered to encode noncanonical amino acids by introducing a newly evolved aminoacyl–tRNA synthetase/tRNA pair in E. coli. The success of this new method provides options for future expansion of the genetic code.
      PubDate: 2016-10-13T08:50:50.385327-05:
      DOI: 10.1002/cbic.201600448
       
  • Exploring the Potent Inhibition of CTP Synthase by
           Gemcitabine-5′-Triphosphate

    • Authors: Gregory D. McCluskey; Samy Mohamady, Scott D. Taylor, Stephen L. Bearne
      Pages: 2240 - 2249
      Abstract: CTP synthase (CTPS) catalyzes the conversion of UTP to CTP and is a target for the development of antiviral, anticancer, antiprotozoal, and immunosuppressive agents. Exposure of cell lines to the antineoplastic cytidine analogue gemcitabine causes depletion of intracellular CTP levels, but the direct inhibition of CTPS by its metabolite gemcitabine-5′-triphosphate (dF-dCTP) has not been demonstrated. We show that dF-dCTP is a potent competitive inhibitor of Escherichia coli CTPS with respect to UTP [Ki=(3.0±0.1) μm], and that its binding affinity exceeds that of CTP ≈75-fold. Site-directed mutagenesis studies indicated that Glu149 is an important binding determinant for both CTP and dF-dCTP. Comparison of the binding affinities of the 5′-triphosphates of 2′-fluoro-2′-deoxycytidine and 2′-fluoro-2′-deoxyarabinocytidine revealed that the 2′-F-arabino group contributes markedly to the strong binding of dF-dCTP. Geminal 2′-F substitution on UTP (dF-dUTP) did not result in an increase in binding affinity with CTPS. Remarkably, CTPS catalyzed the conversion of dF-dUTP into dF-dCTP, thus suggesting that dF-dCTP might be regenerated in vivo from its catabolite dF-dUTP.Fluorinated nucleotides: Gemcitabine-5′-triphosphate, a metabolite of the anticancer drug gemcitabine, is a potent competitive inhibitor of the chemotherapeutic target CTP synthase (CTPS). CTPS can use the corresponding 2′,2′-difluoro derivative of UTP as a substrate, thus furnishing a mechanism for regenerating the active drug in vivo.
      PubDate: 2016-11-16T07:15:23.436979-05:
      DOI: 10.1002/cbic.201600405
       
  • Site-Specific Immobilization of the Peptidoglycan Synthase PBP1B on a
           Surface Plasmon Resonance Chip Surface

    • Authors: Inge L. van't Veer; Nadia O. L. Leloup, Alexander J. F. Egan, Bert J. C. Janssen, Nathaniel I. Martin, Waldemar Vollmer, Eefjan Breukink
      Pages: 2250 - 2256
      Abstract: Surface plasmon resonance (SPR) is one of the most powerful label-free methods to determine the kinetic parameters of molecular interactions in real time and in a highly sensitive way. Penicillin-binding proteins (PBPs) are peptidoglycan synthesis enzymes present in most bacteria. Established protocols to analyze interactions of PBPs by SPR involve immobilization to an ampicillin-coated chip surface (a β-lactam antibiotic mimicking its substrate), thereby forming a covalent complex with the PBPs transpeptidase (TP) active site. However, PBP interactions measured with a substrate-bound TP domain potentially affect interactions near the TPase active site. Furthermore, in vivo PBPs are anchored in the inner membrane by an N-terminal transmembrane helix, and hence immobilization at the C-terminal TPase domain gives an orientation contrary to the in vivo situation. We designed a new procedure: immobilization of PBP by copper-free click chemistry at an azide incorporated in the N terminus. In a proof-of-principle study, we immobilized Escherichia coli PBP1B on an SPR chip surface and used this for the analysis of the well-characterized interaction of PBP1B with LpoB. The site-specific incorporation of the azide affords control over protein orientation, thereby resulting in a homogeneous immobilization on the chip surface. This method can be used to study topology-dependent interactions of any (membrane) protein.Site specific immobilization of PBP1B on an SPR chip surface was achieved by the incorporation of an unnatural amino acid with an azide group in combination with a cyclooctyne-functionalized SPR chip surface. This method can be used for the site-specific and homogeneous immobilization of any protein.
      PubDate: 2016-11-07T04:28:05.885987-05:
      DOI: 10.1002/cbic.201600461
       
  • Expression and Purification of EPHA2 Tyrosine Kinase Domain for
           Crystallographic and NMR Studies

    • Authors: Santosh L. Gande; Krishna Saxena, Sridhar Sreeramulu, Verena Linhard, Denis Kudlinzki, Stephanie Heinzlmeir, Andreas J. Reichert, Arne Skerra, Bernhard Kuster, Harald Schwalbe
      Pages: 2257 - 2263
      Abstract: The receptor tyrosine kinase EPHA2 is overexpressed in several cancers (breast, head and neck, non-small-cell lung cancer). Small-molecule-based inhibition of the EPHA2 kinase domain (KD) is seen as an important strategy for therapeutic intervention. However, obtaining structural information by crystallography or NMR spectroscopy for drug discovery is severely hampered by the lack of pure, homogeneous protein. Here, different fragments of the EPHA2 KD were expressed and purified from both bacterial (Escherichia coli, BL21(DE3) cells) and insect cells (Spodoptera frugiperda, Sf9 cells).1H,15N HSQC was used to determine the proper folding and homogeneity of all the constructs. Protein from E. coli was well-folded but unstable, and it did not crystallize. However, a construct (D596–G900) produced in Sf9 cells yielded homogenous, well-folded protein that crystallized readily, thereby resulting in eleven new EPHA2–ligand crystal structures. We have also established a strategy for selective and uniform 15N-amino acid labeling of EPHA2 KD in Sf9 cells for investigating dynamics and EPHA2–drug interactions by NMR.EPHA2 tyrosine kinase: EPHA2 is overexpressed in several cancers, but structural analysis is hampered by the lack of stable homogeneous protein. An optimized kinase construct was expressed in Sf9 cells to pursue study by NMR and protein crystallography. This work will aid drug discovery and development.
      PubDate: 2016-11-09T15:58:54.878153-05:
      DOI: 10.1002/cbic.201600483
       
  • A Second, Druggable Binding Site in UDP-Galactopyranose Mutase from
           Mycobacterium tuberculosis?

    • Authors: Yun Shi; Cinzia Colombo, Jijin R. A. Kuttiyatveetil, Nataliya Zalatar, Karin E. van Straaten, Sankar Mohan, David A. R. Sanders, B. Mario Pinto
      Pages: 2264 - 2273
      Abstract: UDP-galactopyranose mutase (UGM), a key enzyme in the biosynthesis of mycobacterial cell walls, is a potential target for the treatment of tuberculosis. In this work, we investigate binding models of a non-substrate-like inhibitor, MS-208, with M. tuberculosis UGM. Initial saturation transfer difference (STD) NMR experiments indicated a lack of direct competition between MS-208 and the enzyme substrate, and subsequent kinetic assays showed mixed inhibition. We thus hypothesized that MS-208 binds at an allosteric binding site (A-site) instead of the enzyme active site (S-site). A candidate A-site was identified in a subsequent computational study, and the overall hypothesis was supported by ensuing mutagenesis studies of the A-site. Further molecular dynamics studies led us to propose that MS-208 inhibition occurs by preventing complete closure of an active site mobile loop that is necessary for productive substrate binding. The results suggest the presence of an A-site with potential druggability, opening up new opportunities for the development of novel drug candidates against tuberculosis.Second site: NMR and MD experiments have unveiled a possible second binding site on UDP-galactopyranose mutase (UGM), a target for tuberculosis. The best MD model showed that the investigated inhibitor bound at an allosteric site rather than the substrate-binding site. This was corroborated by experimental results from NMR spectroscopy, kinetic assays, and mutagenesis.
      PubDate: 2016-11-07T04:28:13.39241-05:0
      DOI: 10.1002/cbic.201600469
       
  • Covalent Tethering and Residues with Bulky Hydrophobic Side Chains Enable
           Self-Assembly of Distinct Amyloid Structures

    • Authors: Jérémy Ruiz; Régis Boehringer, Marcel Grogg, Jésus Raya, Alicia Schirer, Corinne Crucifix, Petra Hellwig, Patrick Schultz, Vladimir Torbeev
      Pages: 2274 - 2285
      Abstract: Polymorphism is a common property of amyloid fibers that complicates their detailed structural and functional studies. Here we report experiments illustrating the chemical principles that enable the formation of amyloid polymorphs with distinct stoichiometric composition. Using appropriate covalent tethering we programmed self-assembly of a model peptide corresponding to the [20–41] fragment of human β2-microglobulin into fibers with either trimeric or dimeric amyloid cores. Using a set of biophysical and biochemical methods we demonstrated their distinct structural, morphological, and templating properties. Furthermore, we showed that supramolecular approaches in which the peptide is modified with bulky substituents can also be applied to modulate the formation of different fiber polymorphs. Such strategies, when applied to disease-related peptides and proteins, will greatly help in the evaluation of the biological properties of structurally distinct amyloids.Amyloids under control: covalent tethering and supramolecular strategies were successfully used to direct the self-assembly of distinct polymorphs of amyloid fibers. These approaches are potentially useful for the preparation of homogeneous amyloid samples for structural studies and determining the differences in biological activities of amyloid polymorphs.
      PubDate: 2016-11-09T15:38:39.396474-05:
      DOI: 10.1002/cbic.201600440
       
  • Structure and Substrate Recognition of the Bottromycin Maturation Enzyme
           BotP

    • Authors: Greg Mann; Liujie Huo, Sebastian Adam, Brunello Nardone, Jeremie Vendome, Nicholas James Westwood, Rolf Müller, Jesko Koehnke
      Pages: 2286 - 2292
      Abstract: The bottromycins are a family of highly modified peptide natural products, which display potent antimicrobial activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. Bottromycins have recently been shown to be ribosomally synthesized and post-translationally modified peptides (RiPPs). Unique amongst RiPPs, the precursor peptide BotA contains a C-terminal “follower” sequence, rather than the canonical N-terminal “leader” sequence. We report herein the structural and biochemical characterization of BotP, a leucyl-aminopeptidase-like enzyme from the bottromycin pathway. We demonstrate that BotP is responsible for the removal of the N-terminal methionine from the precursor peptide. Determining the crystal structures of both apo BotP and BotP in complex with Mn2+ allowed us to model a BotP/substrate complex and to rationalize substrate recognition. Our data represent the first step towards targeted compound modification to unlock the full antibiotic potential of bottro- mycin.How does BotP do it? Understanding the biosynthesis of bottromycins will permit biotechnological engineering to unlock their pharmaceutical potential. The structural and biochemical studies of BotP, an essential maturation enzyme in the bottromycin synthetic pathway, are the first to be presented for the bottromycin biosynthetic enzymes.
      PubDate: 2016-10-20T08:00:34.399436-05:
      DOI: 10.1002/cbic.201600406
       
  • Corrigendum: Examination of Potential Exceptions to the F and S
           Biosynthetic Classification of Fused-Ring Aromatic Polyketides

    • Authors: Robert Thomas
      Pages: 2293 - 2293
      PubDate: 2016-11-10T08:00:23.879231-05:
      DOI: 10.1002/cbic.201600586
       
  • Back Cover: Light-Activated Triazabutadienes for the Modification of a
           Viral Surface (ChemBioChem 23/2016)

    • Authors: Stephanie M. Jensen; Flora W. Kimani, John C. Jewett
      Pages: 2294 - 2294
      Abstract: The back cover picture shows the chemical “arming” of dengue virus with a new photo-activatable crosslinker containing a triazabutadiene moiety. Unlike other light-based crosslinking technologies—that proceed through carbene or radical intermediates—photoisomerization of the triazabutadiene scaffold liberates an aryl diazonium ion, which can go on to react with tyrosine residues in close proximity. More information can be found in the communication by J. C. Jewett et al. on page 2216 in Issue 23, 2016 (
      DOI : 10.1002/cbic.201600508).
      PubDate: 2016-11-15T08:13:16.699128-05:
       
 
 
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