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Sökning: L773:1554 8937 OR L773:1554 8929

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1.
  • Altincekic, Nadide, et al. (författare)
  • Targeting the Main Protease (Mpro, nsp5) by Growth of Fragment Scaffolds Exploiting Structure-Based Methodologies
  • 2024
  • Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 19:2, s. 563-574
  • Tidskriftsartikel (refereegranskat)abstract
    • The main protease Mpro, nsp5, of SARS-CoV-2 (SCoV2) is one of its most attractive drug targets. Here, we report primary screening data using nuclear magnetic resonance spectroscopy (NMR) of four different libraries and detailed follow-up synthesis on the promising uracil-containing fragment Z604 derived from these libraries. Z604 shows time-dependent binding. Its inhibitory effect is sensitive to reducing conditions. Starting with Z604, we synthesized and characterized 13 compounds designed by fragment growth strategies. Each compound was characterized by NMR and/or activity assays to investigate their interaction with Mpro. These investigations resulted in the four-armed compound 35b that binds directly to Mpro. 35b could be cocrystallized with Mpro revealing its noncovalent binding mode, which fills all four active site subpockets. Herein, we describe the NMR-derived fragment-to-hit pipeline and its application for the development of promising starting points for inhibitors of the main protease of SCoV2.
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2.
  • Arnaud, Julie, et al. (författare)
  • Reduction of lectin valency drastically changes glycolipid dynamics in membranes but not surface avidity.
  • 2013
  • Ingår i: ACS chemical biology. - : American Chemical Society (ACS). - 1554-8937 .- 1554-8929. ; 8:9, s. 1918-24
  • Tidskriftsartikel (refereegranskat)abstract
    • Multivalency is proposed to play a role in the strong avidity of lectins for glycosylated cell surfaces and also in their ability to affect membrane dynamics by clustering glycosphingolipids. Lectins with modified valency were designed from the β-propeller fold of Ralstonia solanacearum lectin (RSL) that presents six fucose binding sites. After identification of key amino acids by molecular dynamics calculations, two mutants with reduced valency were produced. Isothermal titration calorimetry confirmed the loss of three high affinity binding sites for both mutants. Crystal structures indicated that residual low affinity binding occurred in W76A but not in R17A. The trivalent R17A mutant presented unchanged avidity toward fucosylated surfaces, when compared to hexavalent RSL. However, R17A is not able anymore to induce formation of membrane invaginations on giant unilamellar vesicules, indicating the crucial role of number of binding sites for clustering of glycolipids. In the human lung epithelial cell line H1299, wt-RSL is internalized within seconds whereas the kinetics of R17A uptake is largely delayed. Neolectins with tailored valency are promising tools to study membrane dynamics.
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6.
  • Bjelic, Sinisa, et al. (författare)
  • Computational design of enone-binding proteins with catalytic activity for the Morita-Baylis-Hillman reaction
  • 2013
  • Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 8:4, s. 749-757
  • Tidskriftsartikel (refereegranskat)abstract
    • The Morita-Baylis-Hillman reaction forms a carbon-carbon bond between the α-carbon of a conjugated carbonyl compound and a carbon electrophile. The reaction mechanism involves Michael addition of a nucleophile catalyst at the carbonyl β-carbon, followed by bond formation with the electrophile and catalyst disassociation to release the product. We used Rosetta to design 48 proteins containing active sites predicted to carry out this mechanism, of which two show catalytic activity by mass spectrometry (MS). Substrate labeling measured by MS and site-directed mutagenesis experiments show that the designed active-site residues are responsible for activity, although rate acceleration over background is modest. To characterize the designed proteins, we developed a fluorescence-based screen for intermediate formation in cell lysates, carried out microsecond molecular dynamics simulations, and solved X-ray crystal structures. These data indicate a partially formed active site and suggest several clear avenues for designing more active catalysts.
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7.
  • Bojar, Daniel, et al. (författare)
  • A Useful Guide to Lectin Binding: Machine-Learning Directed Annotation of 57 Unique Lectin Specificities
  • 2022
  • Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 17:11, s. 2993-3012
  • Tidskriftsartikel (refereegranskat)abstract
    • Glycans are critical to every facet of biology and medicine, from viral infections to embryogenesis. Tools to study glycans are rapidly evolving; however, the majority of our knowledge is deeply dependent on binding by glycan binding proteins (e.g., lectins). The specificities of lectins, which are often naturally isolated proteins, have not been well-defined, making it difficult to leverage their full potential for glycan analysis. Herein, we use a combination of machine learning algorithms and expert annotation to define lectin specificity for this important probe set. Our analysis uses comprehensive glycan microarray analysis of commercially available lectins we obtained using version 5.0 of the Consortium for Functional Glycomics glycan microarray (CFGv5). This data set was made public in 2011. We report the creation of this data set and its use in large-scale evaluation of lectin-glycan binding behaviors. Our motif analysis was performed by integrating 68 manually defined glycan features with systematic probing of computational rules for significant binding motifs using mono- and disaccharides and linkages. Combining machine learning with manual annotation, we create a detailed interpretation of glycan-binding specificity for 57 unique lectins, categorized by their major binding motifs: mannose, complex-type N-glycan, O-glycan, fucose, sialic acid and sulfate, GlcNAc and chitin, Gal and LacNAc, and GalNAc. Our work provides fresh insights into the complex binding features of commercially available lectins in current use, providing a critical guide to these important reagents. ©
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8.
  • Bozhanova, Nina G., et al. (författare)
  • Yellow and Orange Fluorescent Proteins with Tryptophan-based Chromophores
  • 2017
  • Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 12:7, s. 1867-1873
  • Tidskriftsartikel (refereegranskat)abstract
    • Rapid development of new microscopy techniques exposed the need for genetically encoded fluorescent tags with special properties. Recent works demonstrated the potential of fluorescent proteins with tryptophan-based chromophores. We applied rational design and random mutagenesis to the monomeric red fluorescent protein FusionRed and found two groups of mutants carrying a tryptophan-based chromophore: with yellow (535 nm) or orange (565 nm) emission. On the basis of the properties of proteins, a model synthetic chromophore, and a computational modeling, we concluded that the presence of a ketone-containing chromophore in different isomeric forms can explain the observed yellow and orange phenotypes.
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9.
  • Bozzola, Tiago, et al. (författare)
  • Sialic Acid Derivatives Inhibit SiaT Transporters and Delay Bacterial Growth
  • 2022
  • Ingår i: Acs Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 17:7, s. 1890-1900
  • Tidskriftsartikel (refereegranskat)abstract
    • Antibiotic resistance is a major worldwide concern, and new drugs with mechanistically novel modes of action are urgently needed. Here, we report the structure-based drug design, synthesis, and evaluation in vitro and in cellular systems of sialic acid derivatives able to inhibit the bacterial sialic acid symporter SiaT. We designed and synthesized 21 sialic acid derivatives and screened their affinity for SiaT by a thermal shift assay and elucidated the inhibitory mechanism through binding thermodynamics, computational methods, and inhibitory kinetic studies. The most potent compounds, which have a 180-fold higher affinity compared to the natural substrate, were tested in bacterial growth assays and indicate bacterial growth delay in methicillin-resistant Staphylococcus aureus. This study represents the first example and a promising lead in developing sialic acid uptake inhibitors as novel antibacterial agents.
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11.
  • Chen, Z., et al. (författare)
  • In-Depth Site-Specific O-Glycosylation Analysis of Glycoproteins and Endogenous Peptides in Cerebrospinal Fluid (CSF) from Healthy Individuals, Mild Cognitive Impairment (MCI), and Alzheimer's Disease (AD) Patients
  • 2022
  • Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 17:11, s. 3059-3068
  • Tidskriftsartikel (refereegranskat)abstract
    • Site-specific O-glycoproteome mapping in complex biological systems provides a molecular basis for understanding the structure-function relationships of glycoproteins and their roles in physiological and pathological processes. Previous O-glycoproteome analysis in cerebrospinal fluid (CSF) focused on sialylated glycoforms, while missing information on other glycosylation types. In order to achieve an unbiased O-glycosylation profile, we developed an integrated strategy combining universal boronic acid enrichment, high-pH fractionation, and electron-transfer and higher-energy collision dissociation (EThcD) for enhanced intact O-glycopeptide analysis. We applied this strategy to analyze the O-glycoproteome in CSF, resulting in the identification of 308 O-glycopeptides from 110 O-glycoproteins, covering both sialylated and nonsialylated glycoforms. To our knowledge, this is the largest data set of O-glycoproteins and O-glycosites reported for CSF to date. We also developed a peptidomics workflow that utilized the EThcD and a three-step database searching strategy for comprehensive PTM analysis of endogenous peptides, including N-glycosylation, O-glycosylation, and other common peptide PTMs. Interestingly, among the 1411 endogenous peptides identified, 89 were O-glycosylated, and only one N-glycosylated peptide was found, indicating that CSF endogenous peptides were predominantly O-glycosylated. Analyses of the O-glycoproteome and endogenous peptidome PTMs were also conducted in the CSF of MCI and AD patients to provide a landscape of glycosylation patterns in different disease states. Our results showed a decreasing trend in fucosylation and an increasing trend of endogenous peptide O-glycosylation, which may play an important role in AD progression. ©
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13.
  • Danelius, Emma, et al. (författare)
  • MicroED as a Powerful Tool for Structure Determination of Macrocyclic Drug Compounds Directly from Their Powder Formulations
  • 2023
  • Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 18:12, s. 2582-2589
  • Tidskriftsartikel (refereegranskat)abstract
    • Macrocycles are important drug leads with many advantages including the ability to target flat and featureless binding sites as well as to act as molecular chameleons and thereby reach intracellular targets. However, due to their complex structures and inherent flexibility, macrocycles are difficult to study structurally, and there are limited structural data available. Herein, we use the cryo-EM method MicroED to determine the novel atomic structures of several macrocycles that have previously resisted structural determination. We show that structures of similar complexity can now be obtained rapidly from nanograms of material and that different conformations of flexible compounds can be derived from the same experiment. These results will have an impact on contemporary drug discovery as well as natural product exploration.
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14.
  • Deiana, Marco, et al. (författare)
  • A Minimalistic Coumarin Turn-On Probe for Selective Recognition of Parallel G-Quadruplex DNA Structures
  • 2021
  • Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 16:8, s. 1365-1376
  • Tidskriftsartikel (refereegranskat)abstract
    • G-quadruplex (G4) DNA structures are widespread in the human genome and are implicated in biologically important processes such as telomere maintenance, gene regulation, and DNA replication. Guanine-rich sequences with potential to form G4 structures are prevalent in the promoter regions of oncogenes, and G4 sites are now considered as attractive targets for anticancer therapies. However, there are very few reports of small “druglike” optical G4 reporters that are easily accessible through one-step synthesis and that are capable of discriminating between different G4 topologies. Here, we present a small water-soluble light-up fluorescent probe that features a minimalistic amidinocoumarin-based molecular scaffold that selectively targets parallel G4 structures over antiparallel and non-G4 structures. We showed that this biocompatible ligand is able to selectively stabilize the G4 template resulting in slower DNA synthesis. By tracking individual DNA molecules, we demonstrated that the G4-stabilizing ligand perturbs DNA replication in cancer cells, resulting in decreased cell viability. Moreover, the fast-cellular entry of the probe enabled detection of nucleolar G4 structures in living cells. Finally, insights gained from the structure–activity relationships of the probe suggest the basis for the recognition of parallel G4s, opening up new avenues for the design of new biocompatible G4-specific small molecules for G4-driven theranostic applications.
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15.
  • Delguste, Martin, et al. (författare)
  • Regulatory Mechanisms of the Mucin-Like Region on Herpes Simplex Virus during Cellular Attachment
  • 2019
  • Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8937 .- 1554-8929. ; 14:3, s. 534-542
  • Tidskriftsartikel (refereegranskat)abstract
    • Mucin-like regions, characterized by a local high density of O-linked glycosylation, are found on the viral envelope glycoproteins of many viruses. Herpes simplex virus type 1 (HSV-1), for example, exhibits a mucin-like region on its glycoprotein gC, a viral protein involved in initial recruitment of the virus to the cell surface via interaction with sulfated glycosaminoglycans. So far, this mucin-like region has been proposed to play a key role in modulating the interactions with cellular glycosaminoglycans, and in particular to promote release of HSV-1 virions from infected cells. However, the molecular mechanisms and the role as a pathogenicity factor remains unclear. Using single virus particle tracking, we show that the mobility of chondroitin sulfate-bound HSV-1 virions is decreased in absence of the mucin-like region. This decrease in mobility correlates with an increase in HSV-1-chondroitin sulfate binding forces as observed using atomic force microscopy-based force spectroscopy. Our data suggest that the mucin-like region modulates virus-glycosaminoglycan interactions by regulating the affinity, type, and number of glycoproteins involved in the virus-glycosaminoglycan interaction. This study therefore presents new evidence for a role of the mucin-like region in balancing the interaction of HSV-1 with glycosaminoglycans and provides further insights into the molecular mechanisms used by the virus to ensure both successful cell entry and release from the infected cell.
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16.
  • Deroo, Stephanie, et al. (författare)
  • Mechanism and Rates of Exchange of L7/L12 between Ribosomes and the Effects of Binding EF-G
  • 2012
  • Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 7:6, s. 1120-1127
  • Tidskriftsartikel (refereegranskat)abstract
    • The ribosomal stalk complex binds and recruits translation factors to the ribosome during protein biosynthesis. In Escherichia coli the stalk is composed of protein L10 and four copies of L7/L12. Despite the crucial role of the stalk, mechanistic details of L7/L12 subunit exchange are not established. By incubating isotopically labeled intact ribosomes with their unlabeled counterparts we monitored the exchange of the labile stalk proteins by recording mass spectra as a function of time. On the basis of kinetic analysis, we proposed a mechanism whereby exchange proceeds via L7/L12 monomers and dimers. We also compared exchange of L7/L12 from free ribosomes with exchange from ribosomes in complex with elongation factor G (EF-G), trapped in the posttranslocational state by fusidic acid. Results showed that binding of EF-G reduces the L7/L12 exchange reaction of monomers by similar to 27% and of dimers by similar to 47% compared with exchange from free ribosomes. This is consistent with a model in which binding of EF-G does not modify interactions between the L7/L12 monomers but rather one of the four monomers, and as a result one of the two dimers, become anchored to the ribosome-EF-G complex preventing their free exchange. Overall therefore our results not only provide mechanistic insight into the exchange of L7/L12 monomers and dimers and the effects of EF-G binding but also have implications for modulating stability in response to environmental and functional stimuli within the cell.
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17.
  • Fridjonsdottir, Elva, et al. (författare)
  • Region-Specific and Age-Dependent Multitarget Effects of Acetylcholinesterase Inhibitor Tacrine on Comprehensive Neurotransmitter Systems
  • 2022
  • Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 17:1, s. 147-158
  • Tidskriftsartikel (refereegranskat)abstract
    • Regional brain distribution and metabolism of neurotransmitters and their response to drug treatment are fundamentally important for understanding the central effects of neuroactive substances. We used matrix-assisted laser desorption/ionization mass spectrometry imaging in combination with multivariate analysis to visualize in anatomical detail metabolic effects of aging and tacrine-mediated acetylcholinesterase inhibition on comprehensive neurotransmitter systems in multiple mouse brain regions of 12-week-old and 14-month-old mice. We detected age-related increases in 3,4-dihydroxyphenylacetaldehyde and histamine, indicating oxidative stress and aging deficits in astrocytes. Tacrine had a significant impact on the metabolism of neurotransmitters in both age groups; predominantly, there was an increased norepinephrine turnover throughout the brain and decreased 3-methoxy tyramine, a marker for dopamine release, in the striatum. The striatal levels of histamine were only elevated after tacrine administration in the older animals. Our results demonstrated that tacrine is a multitarget and region-specific neuroactive agent, inducing age-specific responses. Although well-studied, the complete mechanisms of the action of tacrine are not fully understood, and the current findings reveal features that may help explain its treatment-related effectiveness and central side effects.
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18.
  • Gunasekera, Sunithi, 1977-, et al. (författare)
  • Stabilized Cyclic Peptides as Scavengers of Autoantibodies : Neutralization of Anticitrullinated Protein/Peptide Antibodies in Rheumatoid Arthritis
  • 2018
  • Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 13:6, s. 1525-1535
  • Tidskriftsartikel (refereegranskat)abstract
    • The occurrence of autoantibodies is a hallmark of rheumatoid arthritis, specifically those autoantibodies targeting proteins containing the arginine-derived amino acid citrulline. There is strong evidence showing that the occurrence of anticitrullinated protein/peptide antibodies (ACPA) are involved in disease progression, and ACPA was recently shown to induce pain in animals. Here, we explore a novel concept useful for research, diagnostics, and possibly therapy of autoimmune diseases, namely, to directly target and neutralize autoantibodies using peptide binders. A high-affinity peptide-based scavenger of ACPA was developed by grafting a citrullinated epitope derived from human fibrinogen into a naturally occurring stable peptide scaffold. The best scavenger comprises the truncated epitope α-fibrinogen, [Cit573]fib(566-580), grafted into the scaffold sunflower trypsin inhibitor-1, SFTI-1. The final peptide demonstrates low nanomolar apparent affinity and superior stability.
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19.
  • Hammarstrom, LGJ, et al. (författare)
  • Chemical Biology Consortium Sweden
  • 2013
  • Ingår i: ACS chemical biology. - : American Chemical Society (ACS). - 1554-8937 .- 1554-8929. ; 8:12, s. 2605-2606
  • Tidskriftsartikel (refereegranskat)
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20.
  • Haugaard-Kedström, Linda M., et al. (författare)
  • Solution Structure, Aggregation Behavior, and Flexibility of Human Relaxin-2.
  • 2015
  • Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 10:3, s. 891-900
  • Tidskriftsartikel (refereegranskat)abstract
    • Relaxin is a member of the relaxin/insulin peptide hormone superfamily and is characterized by a two-chain structure constrained by three disulfide bonds. Relaxin is a pleiotropic hormone and involved in a number of physiological and pathogenic processes, including collagen and cardiovascular regulation and tissue remodelling during pregnancy and cancer. Crystallographic and ultracentrifugation experiments have revealed that the human form of relaxin, H2 relaxin, self-associates into dimers, but the significance of this is poorly understood. Here, we present the NMR structure of a monomeric, amidated form of H2 relaxin and compare its features and behavior in solution to those of native H2 relaxin. The overall structure of H2 relaxin is retained in the monomeric form. H2 relaxin amide is fully active at the relaxin receptor RXFP1 and thus dimerization is not required for biological activity. Analysis of NMR chemical shifts and relaxation parameters identified internal motion in H2 relaxin at the pico-nanosecond and milli-microsecond time scales, which is commonly seen in other relaxin and insulin peptides and might be related to function.
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21.
  • Hedberg, Christian, et al. (författare)
  • Molecular perspectives on protein adenylylation
  • 2015
  • Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 10:1, s. 12-21
  • Forskningsöversikt (refereegranskat)abstract
    • In the cell, proteins are frequently modified covalently at specific amino acids with post-translational modifications, leading to a diversification of protein functions and activities. Since the introduction of high-resolution mass spectrometry, new post-translational modifications are constantly being discovered. One particular modification is the adenylylation of mammalian proteins. In adenylylation, adenosine triphosphate (ATP) is utilized to attach an adenosine monophosphate at protein threonine or tyrosine residues via a phosphodiester linkage. Adenylylation is particularly interesting in the context of infections by bacterial pathogens during which mammalian proteins are manipulated through AMP attachment via secreted bacterial factors. In this review, we summarize the role and regulation of enzymatic adenylylation and the mechanisms of catalysis. We also refer to recent methods for the detection of adenylylated proteins by modification-specific antibodies, ATP analogues equipped with chemical handles, and mass spectrometry approaches. Additionally, we review screening approaches for inhibiting adenylylation and briefly discuss related modifications such as phosphocholination and phosphorylation.
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22.
  • Hering, Jenny, et al. (författare)
  • Exploring the Active Site of the Antibacterial Target MraY by Modified Tunicamycins.
  • 2020
  • Ingår i: ACS chemical biology. - : American Chemical Society (ACS). - 1554-8937 .- 1554-8929. ; 15:11, s. 2885-2895
  • Tidskriftsartikel (refereegranskat)abstract
    • The alarming growth of antibiotic resistance that is currently ongoing is a serious threat to human health. One of the most promising novel antibiotic targets is MraY (phospho-MurNAc-pentapeptide-transferase), an essential enzyme in bacterial cell wall synthesis. Through recent advances in biochemical research, there is now structural information available for MraY, and for its human homologue GPT (GlcNAc-1-P-transferase), that opens up exciting possibilities for structure-based drug design. The antibiotic compound tunicamycin is a natural product inhibitor of MraY that is also toxic to eukaryotes through its binding to GPT. In this work, we have used tunicamycin and modified versions of tunicamycin as tool compounds to explore the active site of MraY and to gain further insight into what determines inhibitor potency. We have investigated tunicamycin variants where the following motifs have been modified: the length and branching of the tunicamycin fatty acyl chain, the saturation of the fatty acyl chain, the 6″-hydroxyl group of the GlcNAc ring, and the ring structure of the uracil motif. The compounds are analyzed in terms of how potently they bind to MraY, inhibit the activity of the enzyme, and affect the protein thermal stability. Finally, we rationalize these results in the context of the protein structures of MraY and GPT.
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23.
  • Huang, Xiaoli, et al. (författare)
  • Semisynthesis of SY-1 for Investigation of Breast Cancer Stem Cell Selectivity of C-Ring-Modified Salinomycin Analogues.
  • 2014
  • Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8937 .- 1554-8929. ; 9:7, s. 1587-1594
  • Tidskriftsartikel (refereegranskat)abstract
    • Salinomycin, a naturally occurring polyether ionophore was recently found to selectively reduce the proportion of CD44(+)/CD24(-) cells, a phenotype associated with breast cancer stem cells. Subsequent studies from our group showed that chemical modification of the allylic C20 hydroxyl of salinomycin, located at the C-ring, can enhance the activity of derivatives against breast cancer cells over 5-fold compared to the native structure. Access to C-ring-modified salinomycin analogues is thus of interest from both a mechanistic and a synthetic perspective. Here, we report efficient strategies for gram scale synthesis of the natural product SY-1 (20-deoxy salinomycin), and a saturated analogue, 18,19-dihydro SY-1, for a comparative in vitro investigation of the biological profiles of these compounds with that of salinomycin. Across several assays, the deoxygenated structures required higher concentrations to elicit similar cellular responses to that of salinomycin. Similarly to salinomycin, SY-1 or 18,19-dihydro SY-1 treatment was found to reduce the proportion of CD44(+)/CD24(-) cells with essentially complete selectivity up to ∼IC25. Importantly, the proportion of CD44(+)/CD24(-) cells showed a pronounced U-shaped dose response curve for salinomycin and its derivatives, but not for paclitaxel. The concentration for maximum response in this assay followed differences in IC50 for salinomycin and its analogues, which emphasizes the importance of taking concentration dependence into account when comparing effects on the CD44(+)/CD24(-) phenotype. Small differences in the global conformation within the triad of compounds investigated together with differences in activity across assays emphasize the importance of substitution at C20 for the activity of salinomycin and its derivatives.
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24.
  • Jensen, Thomas M. T., et al. (författare)
  • Molecular Details of a Coupled Binding and Folding Reaction between the Amyloid Precursor Protein and a Folded Domain
  • 2021
  • Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 16:7, s. 1191-1200
  • Tidskriftsartikel (refereegranskat)abstract
    • Intrinsically disordered regions in proteins often function as binding motifs in protein-protein interactions. The mechanistic aspects and molecular details of such coupled binding and folding reactions, which involve formation of multiple noncovalent bonds, have been broadly studied theoretically, but experimental data are scarce. Here, using a combination of protein semisynthesis to incorporate phosphorylated amino acids, backbone amide-to-ester modifications, side chain substitutions, and binding kinetics, we examined the interaction between the intrinsically disordered motif of amyloid precursor protein (APP) and the phosphotyrosine binding (PTB) domain of Mint2. We show that the interaction is regulated by a self-inhibitory segment of the PTB domain previously termed ARM. The helical ARM linker decreases the association rate constant 30-fold through a fast pre-equilibrium between an open and a closed state. Extensive side chain substitutions combined with kinetic experiments demonstrate that the rate-limiting transition state for the binding reaction is governed by native and non-native hydrophobic interactions and hydrogen bonds. Hydrophobic interactions were found to be particularly important during crossing of the transition state barrier. Furthermore, linear free energy relationships show that the overall coupled binding and folding reaction involves cooperative formation of interactions with roughly 30% native contacts formed at the transition state. Our data support an emerging picture of coupled binding and folding reactions following overall chemical principles similar to those of folding of globular protein domains but with greater malleability of ground and transition states.
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25.
  • Johansson, Emil, et al. (författare)
  • Pentavalent Sialic Acid Conjugates Block Coxsackievirus A24 Variant and Human Adenovirus Type 37-Viruses That Cause Highly Contagious Eye Infections
  • 2020
  • Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 15:10, s. 2683-2691
  • Tidskriftsartikel (refereegranskat)abstract
    • Coxsackievirus A24 variant (CVA24v) and human adenovirus 37 (HAdV-37) are leading causative agents of the severe and highly contagious ocular infections acute hemorrhagic conjunctivitis and epidemic keratoconjunctivitis, respectively. Currently, neither vaccines nor antiviral agents are available for treating these diseases, which affect millions of individuals worldwide. CVA24v and HAdV-37 utilize sialic acid as attachment receptors facilitating entry into host cells. Previously, we and others have shown that derivatives based on sialic acid are effective in preventing HAdV-37 binding and infection of cells. Here, we designed and synthesized novel pentavalent sialic acid conjugates and studied their inhibitory effect against CVA24v and HAdV-37 binding and infection of human corneal epithelial cells. The pentavalent conjugates are the first reported inhibitors of CVA24v infection and proved efficient in blocking HAdV-37 binding. Taken together, the pentavalent conjugates presented here form a basis for the development of general inhibitors of these highly contagious ocular pathogens.
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26.
  • Kampen, Stefanie, et al. (författare)
  • Structure-Based Discovery of Negative Allosteric Modulators of the Metabotropic Glutamate Receptor 5
  • 2022
  • Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 17:10, s. 2744-2752
  • Tidskriftsartikel (refereegranskat)abstract
    • Recently determined structures of class C G protein-coupled receptors (GPCRs) revealed the location of allosteric binding sites and opened new opportunities for the discovery of novel modulators. In this work, molecular docking screens for allosteric modulators targeting the metabotropic glutamate receptor 5 (mGlu(5)) were performed. The mGlu(5) receptor is activated by the main excitatory neurotransmitter of the nervous central system, L-glutamate, and mGlu(5) receptor activity can be allosterically modulated by negative or positive allosteric modulators. The mGlu(5) receptor is a promising target for the treatment of psychiatric and neurodegenerative diseases, and several allosteric modulators of this GPCR have been evaluated in clinical trials. Chemical libraries containing fragment-(1.6 million molecules) and lead-like (4.6 million molecules) compounds were docked to an allosteric binding site of mGlu(5) identified in X-ray crystal structures. Among the top-ranked compounds, 59 fragments and 59 lead-like compounds were selected for experimental evaluation. Of these, four fragment-and seven lead-like compounds were confirmed to bind to the allosteric site with affinities ranging from 0.43 to 8.6 mu M, corresponding to a hit rate of 9%. The four compounds with the highest affinities were demonstrated to be negative allosteric modulators of mGlu(5) signaling in functional assays. The results demonstrate that virtual screens of fragment and lead-like chemical libraries have complementary advantages and illustrate how access to high-resolution structures of GPCRs in complex with allosteric modulators can accelerate lead discovery.
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27.
  • Kao, Mu-Rong, et al. (författare)
  • A Robust α-L-Fucosidase from Prevotella nigrescens for Glycoengineering Therapeutic Antibodies
  • 2024
  • Ingår i: ACS Chemical Biology. - 1554-8929 .- 1554-8937. ; 19:7, s. 1515-1524
  • Tidskriftsartikel (refereegranskat)abstract
    • Eliminating the core fucose from the N-glycans of the Fc antibody segment by pathway engineering or enzymatic methods has been shown to enhance the potency of therapeutic antibodies, especially in the context of antibody-dependent cytotoxicity (ADCC). However, there is a significant challenge due to the limited defucosylation efficiency of commercially available α-l-fucosidases. In this study, we report a unique α-l-fucosidase (PnfucA) from the bacterium Prevotella nigrescens that has a low sequence identity compared with all other known α-l-fucosidases and is highly reactive toward a core disaccharide substrate with fucose α(1,3)-, α (1,4)-and α(1,6)-linked to GlcNAc, and is less reactive toward the Fuc-α(1,2)-Gal on the terminal trisaccharide of the oligosaccharide Globo H (Bb3). The kinetic properties of the enzyme, such as its Km and kcat, were determined and the optimized expression of PnfucA gave a yield exceeding 30 mg/L. The recombinant enzyme retained its full activity even after being incubated for 6 h at 37 °C. Moreover, it retained 92 and 87% of its activity after freezing and freeze-drying treatments, respectively, for over 28 days. In a representative glycoengineering of adalimumab (Humira), PnfucA showed remarkable hydrolytic efficiency in cleaving the α(1,6)-linked core fucose from FucGlcNAc on the antibody with a quantitative yield. This enabled the seamless incorporation of biantennary sialylglycans by Endo-S2 D184 M in a one-pot fashion to yield adalimumab in a homogeneous afucosylated glycoform with an improved binding affinity toward Fcγ receptor IIIa.
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28.
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29.
  • Kuru, Erkin, et al. (författare)
  • Mechanisms of Incorporation for D-Amino Acid Probes That Target Peptidoglycan Biosynthesis
  • 2019
  • Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 14:12, s. 2745-2756
  • Tidskriftsartikel (refereegranskat)abstract
    • Bacteria exhibit a myriad of different morphologies, through the synthesis and modification of their essential peptidoglycan (PG) cell wall. Our discovery of a fluorescent D-amino acid (FDAA)-based PG labeling approach provided a powerful method for observing how these morphological changes occur. Given that PG is unique to bacterial cells and a common target for antibiotics, understanding the precise mechanism(s) for incorporation of (F)DAA-based probes is a crucial determinant in understanding the role of PG synthesis in bacterial cell biology and could provide a valuable tool in the development of new antimicrobials to treat drug-resistant antibacterial infections. Here, we systematically investigate the mechanisms of FDAA probe incorporation into PG using two model organisms Escherichia coli (Gram-negative) and Bacillus subtilis (Gram-positive). Our in vitro and in vivo data unequivocally demonstrate that these bacteria incorporate FDAAs using two extracytoplasmic pathways: through activity of their D,D-transpeptidases, and, if present, by their L,D-transpeptidases and not via cytoplasmic incorporation into a D-Ala-D-Ala dipeptide precursor. Our data also revealed the unprecedented finding that the DAA-drug, D-cycloserine, can be incorporated into peptide stems by each of these transpeptidases, in addition to its known inhibitory activity against D-alanine racemase and D-Ala-D-Ala ligase. These mechanistic findings enabled development of a new, FDAA-based, in vitro labeling approach that reports on subcellular distribution of muropeptides, an especially important attribute to enable the study of bacteria with poorly defined growth modes. An improved understanding of the incorporation mechanisms utilized by DAA-based probes is essential when interpreting results from high resolution experiments and highlights the antimicrobial potential of synthetic DAAs.
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30.
  • Larsson, Karl-Magnus, et al. (författare)
  • Structural Basis for Adenosylcobalamin Activation in AdoCbl-Dependent Ribonucleotide Reductases
  • 2010
  • Ingår i: ACS chemical biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 5:10, s. 933-942
  • Tidskriftsartikel (refereegranskat)abstract
    • Class II ribonucleotide reductases (RNR) catalyze the formation of an essential thiyl radical by homolytic cleavage of the Co-C bond in their adenosylcobalamin (AdoCbl) cofactor. Several mechanisms for the dramatic acceleration of Co-C bond cleavage in AdoCbl-dependent enzymes have been advanced, but no consensus yet exists. We present the structure of the class II RNR from Thermo toga maritima in three complexes: (i) with allosteric effector dTTP, substrate GDP, and AdoCbl; (ii) with dTTP and AdoCbl; (iii) with dTTP, GDP, and adenosine. Comparison of these structures gives the deepest structural insights so far into the mechanism of radical generation and transfer for AdoCbl-dependent RNR. AdoCbl binds to the active site pocket, shielding the substrate, transient 5'-deoxyadenosyl radical and nascent thiyl radical from solution. The e-propionamide side chain of AdoCbl forms hydrogen bonds directly to the alpha-phosphate group of the substrate. This interaction appears to cause a "locking-in" of the cofactor, and it is the first observation of a direct cofactor-substrate interaction in an AdoCbl-dependent enzyme. The structures support an ordered sequential reaction mechanism with release or relaxation of AdoCbl on each catalytic cycle. A conformational change of the AdoCbl adenosyl ribose is required to allow hydrogen transfer to the catalytic thiol group. Previously proposed Mechanisms for radical transfer in B12-dependent enzymes cannot fully explain the transfer in class II RNR, suggesting that it may form a separate class that differs from the well characterized eliminases and mutases.
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31.
  • Liljas, Anders (författare)
  • Deepening ribosomal insights
  • 2006
  • Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8937 .- 1554-8929. ; 1:9, s. 567-569
  • Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
    • The remarkable progress of cryoelectron microscopy and crystallography in elucidating ribosomal structure and function continues. Most recently, two papers about complete 70S ribosomes from Thermus thermophilus at 2.8- and 3.7-angstrom resolution give us more details about the conformations of bound transfer RNA (tRNA) molecules; the bridges between subunits; the locations and roles of proteins, magnesium ions, and water molecules; and the dynamics of ribosomes. Very significant new insights have been gained, particularly for the tRNAs, which can only be studied in their entirety in full ribosomes.
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32.
  • Liljeruhm, Josefine, et al. (författare)
  • Kinetics of D-amino acid incorporation in translation
  • 2019
  • Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 14:2, s. 204-213
  • Tidskriftsartikel (refereegranskat)abstract
    • Despite the stereospecificity of translation for l-amino acids (l-AAs) in vivo, synthetic biologists have enabled ribosomal incorporation of d-AAs in vitro toward encoding polypeptides with pharmacologically desirable properties. However, the steps in translation limiting d-AA incorporation need clarification. In this work, we compared d- and l-Phe incorporation in translation by quench-flow kinetics, measuring 250-fold slower incorporation into the dipeptide for the d isomer from a tRNAPhe-based adaptor (tRNAPheB). Incorporation was moderately hastened by tRNA body swaps and higher EF-Tu concentrations, indicating that binding by EF-Tu can be rate-limiting. However, from tRNAAlaB with a saturating concentration of EF-Tu, the slow d-Phe incorporation was unexpectedly very efficient in competition with incorporation of the l isomer, indicating fast binding to EF-Tu, fast binding of the resulting complex to the ribosome, and rate-limiting accommodation/peptide bond formation. Subsequent elongation with an l-AA was confirmed to be very slow and inefficient. This understanding helps rationalize incorporation efficiencies in vitro and stereospecific mechanisms in vivo and suggests approaches for improving incorporation.
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33.
  • Lindgren, Anders E. G., et al. (författare)
  • PARP Inhibitor with Selectivity Toward ADP-Ribosyltransferase ARTD3/PARP3
  • 2013
  • Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 8:8, s. 1698-1703
  • Tidskriftsartikel (refereegranskat)abstract
    • Inhibiting ADP-ribosyl transferases with PARP-inhibitors is considered a promising strategy for the treatment of many cancers and ischemia, but most of the cellular targets are poorly characterized. Here, we describe an inhibitor of ADP-ribosyltransferase-3/poly(ADP-ribose) polymerase-3 (ARTD3), a regulator of DNA repair and mitotic progression. In vitro profiling against 12, members of the enzyme family suggests selectivity for ARTD3, and crystal structures illustrate the molecular basis for inhibitor selectivity. The compound is active in cells, where it elicits ARTD3-specific effects at submicromolar concentration. Our results show that by targeting the nicotinamide binding site, selective inhibition can be achieved among the closest relatives of the validated clinical target, ADP-ribosyltransferase-1/poly(ADP-ribose) polymerase-1.
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34.
  • Lindström, Ida, et al. (författare)
  • Dynamics, Conformational Entropy, and Frustration in Protein-Protein Interactions Involving an Intrinsically Disordered Protein Domain
  • 2018
  • Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 13:5, s. 1218-1227
  • Tidskriftsartikel (refereegranskat)abstract
    • Intrinsically disordered proteins (IDPs) are abundant in the eukaryotic proteome. However, little is known about the role of subnanosecond dynamics and the conformational entropy that it represents in protein-protein interactions involving IDPs. Using nuclear magnetic resonance side chain and backbone relaxation, stopped-flow kinetics, isothermal titration calorimetry, and computational studies, we have characterized the interaction between the globular TAZ1 domain of the CREB binding protein and the intrinsically disordered transactivation domain of STAT2 (TAD-STAT2). We show that the TAZ1/TAD-STAT2 complex retains considerable subnanosecond motions, with TAD-STAT2 undergoing only a partial disorder-to-order transition. We report here the first experimental determination of the conformational entropy change for both binding partners in an IDP binding interaction and find that the total change even exceeds in magnitude the binding enthalpy and is comparable to the contribution from the hydrophobic effect, demonstrating its importance in the binding energetics. Furthermore, we show that the conformational entropy change for TAZ1 is also instrumental in maintaining a biologically meaningful binding affinity. Strikingly, a spatial clustering of very high amplitude motions and a cluster of more rigid sites in the complex exist, which through computational studies we found to overlap with regions that experience energetic frustration and are less frustrated, respectively. Thus, the residual dynamics in the bound state could be necessary for faster dissociation, which is important for proteins that interact with multiple binding partners.
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35.
  • Mannel, Barbara, et al. (författare)
  • Structure-Guided Screening for Functionally Selective D-2 Dopamine Receptor Ligands from a Virtual Chemical Library
  • 2017
  • Ingår i: ACS Chemical Biology. - : AMER CHEMICAL SOC. - 1554-8929 .- 1554-8937. ; 12:10, s. 2652-2661
  • Tidskriftsartikel (refereegranskat)abstract
    • Functionally selective ligands stabilize conformations of G protein-coupled receptors (GPCRs) that induce a preference for signaling via a subset of the intracellular pathways activated by the endogenous agonists. The possibility to fine-tune the functional activity of a receptor provides opportunities to develop drugs that selectively signal via pathways associated with a therapeutic effect and avoid those causing side effects. Animal studies have indicated that ligands displaying functional selectivity at the D-2 dopamine receptor (D2R) could be safer and more efficacious drugs against neuropsychiatric diseases. In this work, computational design of functionally selective D2R ligands was explored using structure-based virtual screening. Molecular docking of known functionally selective ligands to a D2R homology model indicated that such compounds were anchored by interactions with the orthosteric site and extended into a common secondary pocket. A tailored virtual library with close to 13-000 compounds bearing 2,3-dichlorophenylpiperazine, a privileged orthosteric scaffold, connected to diverse chemical moieties via a linker was docked to the D2R model. Eighteen top-ranked compounds that occupied both the orthosteric and allosteric site were synthesized, leading to the discovery of 16 partial agonists. A majority of the ligands had comparable maximum effects in the G protein and beta-arrestin recruitment assays, but a subset displayed preference for a single pathway. In particular, compound 4 stimulated beta-arrestin recruitment (EC50 = 320 nM, E-max = 16%) but had no detectable G protein signaling. The use of structure-based screening and virtual libraries to discover GPCR ligands with tailored functional properties will be discussed.
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36.
  • McGeachie, Andrew B, et al. (författare)
  • Pyrimidyn Compounds : Dual-Action Small Molecule Pyrimidine-Based Dynamin Inhibitors
  • 2013
  • Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 8:7, s. 1507-1518
  • Tidskriftsartikel (refereegranskat)abstract
    • Dynamin is required for clathrin-mediated endocytosis (CME). Its GTPase activity is stimulated by phospholipid binding to its PH domain, which induces helical oligomerization. We have designed a series of novel pyrimidine-based "Pyrimidyn" compounds that inhibit the lipid-stimulated GTPase activity of full length dynamin I and II with similar potency. The most potent analogue, Pyrimidyn 7, has an IC50 of 1.1 μM for dynamin I and 1.8 μM for dynamin II, making it among the most potent dynamin inhibitors identified to date. We investigated the mechanism of action of the Pyrimidyn compounds in detail by examining the kinetics of Pyrimidyn 7 inhibition of dynamin. The compound competitively inhibits both GTP and phospholipid interactions with dynamin I. While both mechanisms of action have been previously observed separately, this is the first inhibitor series to incorporate both and thereby to target two distinct domains of dynamin. Pyrimidyn 6 and 7 reversibly inhibit CME of both transferrin and EGF in a number of non-neuronal cell lines as well as inhibiting synaptic vesicle endocytosis (SVE) in nerve terminals. Therefore, Pyrimidyn compounds block endocytosis by directly competing with GTP and lipid binding to dynamin, limiting both the recruitment of dynamin to membranes and its activation. This dual mode of action provides an important new tool for molecular dissection of dynamin's role in endocytosis.
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37.
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38.
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39.
  • Mojr, Viktor, et al. (författare)
  • Nonhydrolysable Analogues of (p)ppGpp and (p)ppApp Alarmone Nucleotides as Novel Molecular Tools
  • 2021
  • Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 16:9, s. 1680-1691
  • Tidskriftsartikel (refereegranskat)abstract
    • While alarmone nudeotides guanosine-3',5'-bisdiphosphate (ppGpp) and guanosine-5'-triphosphate-3'-diphosphate (pppGpp) are archetypical bacterial second messengers, their adenosine analogues ppApp (adenosine-3',5'-bisdiphosphate) and pppApp (adenosine-5'-triphosphate-3'-diphosphate) are toxic effectors that abrogate bacterial growth. The alarmones are both synthesized and degraded by the members of the ReIA-SpoT Homologue (RSH) enzyme family. Because of the chemical and enzymatic liability of (p)ppGpp and (p)ppApp, these alarmones are prone to degradation during structural biology experiments. To overcome this limitation, we have established an efficient and straightforward procedure for synthesizing nonhydrolysable (p)ppNu(N)pp analogues starting from 3'-azido-3'-deoxyribonucleotides as key intermediates. To demonstrate the utility of (p)ppG(N)pp as a molecular tool, we show that (i) as an HD substrate mimic, ppG(N)pp competes with ppGpp to inhibit the enzymatic activity of human MESHI Small Alarmone Hyrolase, SAH; and (ii) mimicking the allosteric effects of (p)ppGpp, (p)ppG(N)pp acts as a positive regulator of the synthetase activity of long ribosome-associated RSHs Rel and ReIA. Finally, by solving the structure of the N-terminal domain region (NTD) of T. thermophilus Rel complexed with pppG(N)pp, we show that as an HD substrate mimic, the analogue serves as a bona fide orthosteric regulator that promotes the same intra-NTD structural rearrangements as the native substrate.
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40.
  • Nasir, Waqas, et al. (författare)
  • Histo-Blood Group Antigen Presentation Is Critical for Binding of Norovirus VLP to Glycosphingolipids in Model Membranes
  • 2017
  • Ingår i: Acs Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 12:5, s. 1288-1296
  • Tidskriftsartikel (refereegranskat)abstract
    • Virus entry depends on biomolecular recognition at the surface of cell membranes. In the case of glycolipid receptors, these events are expected to be influenced by how the glycan epitope close to the membrane is presented to the virus. This presentation of membrane associated glycans is more restricted than that of glycans in solution, particularly because of orientational constraints imposed on the glycolipid through its lateral interactions with other membrane lipids and proteins. We have developed and employed a total internal reflection fluorescence microscopy-based binding assay and. a scheme for molecular dynamics (MD) membrane simulations to investigate the consequences of various glycan presentation effects. The system studied was histo-blood group antigen (HBGA) epitopes of membrane-bound glycosphingolipids (GSLs) derived from small intestinal epithelium of humans (type 1 chain) and dogs (type 2 chain) interacting with GII.4 norovirus-like particles. Our experimental results showed strong binding to all lipid-linked type 1 chain HBGAs but no or only weak binding to the corresponding type 2 chain HBGAs. This is in contrast to results derived from STD experiments with free HBGAs in solution where binding was observed for Lewis x. The MD data suggest that the strong binding to type 1 chain glycolipids was due to the well-exposed (1,2)-linked alpha-L-Fucp and (1,4)- linked alpha-L-Fucp residues, while the weaker binding or lack of binding to type 2 chain HBGAs was due to the very restricted accessibility of the (1,3) -linked alpha-L-Fucp residue when the glycolipid is embedded in a phospholipid membrane. Our results not only contribute to a general understanding of protein carbohydrate interactions on model membrane surfaces, particularly in the context of virus binding, but also suggest a possible role of human intestinal GSLs as potential receptors for norovirus uptake.
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41.
  • Nguyen, Thao K. N., et al. (författare)
  • Dimerized Glycosaminoglycan Chains Increase FGF Signaling during Zebrafish Development
  • 2013
  • Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 8:5, s. 939-948
  • Tidskriftsartikel (refereegranskat)abstract
    • Proteoglycans (PGs) modulate numerous signaling pathways during development through binding of their glycosaminoglycan (GAG) side chains to various signaling molecules, including fibroblast growth factors (FGFs). A majority of PGs possess two or more GAG side chains, suggesting that GAG multivalency is imperative for biological functions in vivo. However, only a few studies have examined the biological significance of GAG multivalency. In this report, we utilized a library of bis- and tris-xylosides that produce two and three GAG chains on the same scaffold, respectively, thus mimicking PGs, to examine the importance of GAG valency and chain type in regulating FGF/FGFR interactions in vivo in zebrafish. A number of bis- and tris-xylosides, but not mono-xylosides, caused an elongation phenotype upon their injection into embryos. In situ hybridization showed that elongated embryos have elevated expression of the FGF target gene mkp3 but unchanged expression of reporters for other pathways, indicating that FGF/FGFR signaling was specifically hyperactivated. In support of this observation, elongation can be reversed by the tyrosine kinase inhibitor SU5402, mRNA for the FGFR antagonist sprouty4, or FGF8 morpholino. Endogenous GAGs seem to be unaffected after xyloside treatment, suggesting that this is a gain-of-function phenotype. Furthermore, expression of a multivalent but not a monovalent GAG containing syndecan-1 proteoglycan recapitulates the elongation phenotype observed with the bivalent xylosides. On the basis of these in vivo findings, we propose a new model for GAG/FGF/FGFR interactions in which dimerized GAG chains can activate FGF-mediated signal transduction pathways.
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42.
  • Nilsson, K. Peter R., et al. (författare)
  • Imaging distinct conformational states of amyloid-beta fibrils in Alzheimer's disease using novel luminescent probes
  • 2007
  • Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 2:8, s. 553-560
  • Tidskriftsartikel (refereegranskat)abstract
    • Using luminescent conjugated polyelectrolyte probes (LCPs), we demonstrate the possibility to distinguish amyloid-β 1–42 peptide (Aβ1–42) fibril conformations, by analyzing in vitro generated amyloid fibrils of Aβ1–42 formed under quiescent and agitated conditions. LCPs were then shown to resolve such conformational heterogeneity of amyloid deposits in vivo. A diversity of amyloid deposits depending upon morphology and anatomic location was illustrated with LCPs in frozen ex vivo brain sections from a transgenic mouse model (tg-APP swe) of Alzheimer’s disease. Comparative LCP fluorescence showed that compact-core plaques of amyloid β precursor protein transgenic mice were composed of rigid dense amyloid. A more abundant form of amyloid plaque displayed morphology of a compact center with a protruding diffuse exterior. Surprisingly, the compact center of these plaques showed disordered conformations of the fibrils, and the exterior was composed of rigid amyloid protruding from the disordered center. This type of plaque appears to grow from more loosely assembled regions toward solidified amyloid tentacles. This work demonstrates how application of LCPs can prove helpful to monitor aggregate structure of in vivo formed amyloid deposits such as architecture, maturity, and origin.
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43.
  • Nilsson, Peter, et al. (författare)
  • Imaging distinct conformational states of amyloid-β fibrils in Alzheimer's disease using novel luminescent probes
  • 2007
  • Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 2:8, s. 553-560
  • Tidskriftsartikel (refereegranskat)abstract
    • Using luminescent conjugated polyelectrolyte probes (LCPs), we demonstrate the possibility to distinguish amyloid-β 1-42 peptide (Aβ1-42) fibril conformations, by analyzing in vitro generated amyloid fibrils of Aβ1-42 formed under quiescent and agitated conditions. LCPs were then shown to resolve such conformational heterogeneity of amyloid deposits in vivo. A diversity of amyloid deposits depending upon morphology and anatomic location was illustrated with LCPs in frozen ex vivo brain sections from a transgenic mouse model (tg-APPswe) of Alzheimer's disease. Comparative LCP fluorescence showed that compact-core plaques of amyloid β precursor protein transgenic mice were composed of rigid dense amyloid. A more abundant form of amyloid plaque displayed morphology of a compact center with a protruding diffuse exterior. Surprisingly, the compact center of these plaques showed disordered conformations of the fibrils, and the exterior was composed of rigid amyloid protruding from the disordered center. This type of plaque appears to grow from more loosely assembled regions toward solidified amyloid tentacles. This work demonstrates how application of LCPs can prove helpful to monitor aggregate structure of in vivo formed amyloid deposits such as architecture, maturity, and origin.
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44.
  • Nord, Christina, et al. (författare)
  • Isopedopeptins A-H : Cationic Cyclic Lipodepsipeptides from Pedobacter cryoconitis UP508 Targeting WHO Top-Priority Carbapenem-Resistant Bacteria
  • 2020
  • Ingår i: ACS Chemical Biology. - : AMER CHEMICAL SOC. - 1554-8929 .- 1554-8937. ; 15:11, s. 2937-2944
  • Tidskriftsartikel (refereegranskat)abstract
    • Pedobacter cryoconitis strain UP508 was isolated from a soil sample using a mixture of ampicillin, kanamycin, and nalidixic acid for selection. UP508 was found to produce >30 unknown antibacterial peptides, of which eight, isopedopeptins A-H (1-8), were isolated by bioassay-guided fractionation and characterized with respect to structures and biological properties. Compounds 1-8 were all composed of nine amino acid residues and one 3-hydroxy fatty acid residue, and the structures were ring-closed via an ester bond from the C-terminal aspartic acid to the 3-hydroxy fatty acid. The differences between the peptides were the size and branching of the 3-hydroxy fatty acid and the presence of a valine or a 3-hydroxyvaline residue. The isopedopeptins mainly had activity against Gram-negative bacteria, and isopedopeptin B (2), which had the best combination of antibacterial activity, in vitro cytotoxicity, and hemolytic properties, was selected for further studies against a larger panel of Gram-negative bacteria. Isopedopeptin B was found to have good activity against strains of WHO top-priority Gram-negative bacteria, i.e., carbapenem-resistant Acinetobacter baumannii, Escherichia coli, and Pseudomonas aeruginosa, with minimal inhibitory concentrations (MIC) down to 1, 2, and 4 mu g/mL, respectively. Furthermore, compound 2 had activity against colistin-resistant strains of A. baumannii, E. coli, and Klebsiella pneumoniae, with a MIC down to 8, 2, and 4 mu g/mL, respectively. Compound 6 was tested in an E. coli liposome system where it induced significant leakage, indicating membrane disruption as one mechanism involved in isopedopeptin antibacterial activity. Isopedopeptin B stands out as a promising candidate for further studies with the goal to develop a new antibiotic drug.
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45.
  • Nyström, Sofie, et al. (författare)
  • Evidence for Age-Dependent in Vivo Conformational Rearrangement within A beta Amyloid Deposits
  • 2013
  • Ingår i: ACS Chemical Biology. - : American Chemical Society. - 1554-8929 .- 1554-8937. ; 8:6, s. 1128-1133
  • Tidskriftsartikel (refereegranskat)abstract
    • Deposition of aggregated A beta peptide in the brain is one of the major hallmarks of Alzheimers disease. Using a combination of two structurally different, but related, hypersensitive fluorescent amyloid markers, LCOs, reporting on separate ultrastructural elements, we show that conformational rearrangement occurs within A beta plaques of transgenic mouse models as the animals age. This important mechanistic insight should aid the design and evaluation of experiments currently using plaque load as readout.
  •  
46.
  • Pally, Dharma, et al. (författare)
  • Galectin-9 Signaling Drives Breast Cancer Invasion through Extracellular Matrix
  • 2022
  • Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8937 .- 1554-8929. ; 17:6, s. 1376-1386
  • Tidskriftsartikel (refereegranskat)abstract
    • Aberrations in glycan and lectin expression and function represent one of the earliest hallmarks of cancer. Among galectins, a conserved family of β-galactoside-binding lectins, the role of Galectin-9 in immune-tumor interactions is well-established, although its effect on cancer cell behavior remains unclear. In this study, we assayed for, and observed, an association between Galectin-9 expression and invasiveness of breast cancer cells in vitro and in vivo. Genetic perturbation and pharmacological inhibition using novel cognate inhibitors confirmed a positive correlation between Galectin-9 levels and the adhesion of invasive cancer cells to─and their invasion through─constituted organomimetic extracellular matrix microenvironments. Signaling experiments and unbiased quantitative proteomics revealed Galectin-9 induction of Focal Adhesion Kinase activity and S100A4 expression, respectively. FAK inhibition decreased S100A4 mRNA levels. Our results provide crucial insights into how elevated Galectin-9 expression potentiates the invasiveness of breast cancer cells during early steps of invasion.
  •  
47.
  • Pansieri, Jonathan, et al. (författare)
  • Pro-Inflammatory S100A9 Protein Aggregation Promoted by NCAM1 Peptide Constructs
  • 2019
  • Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 14:7, s. 1410-1417
  • Tidskriftsartikel (refereegranskat)abstract
    • Amyloid cascade and neuroinflammation are hallmarks of neurodegenerative diseases, and pro-inflammatory S100A9 protein is central to both of them. Here, we have shown that NCAM1 peptide constructs carrying polycationic sequences derived from A beta peptide (KKLVFF) and PrP protein (KKRPKP) significantly promote the S100A9 amyloid self-assembly in a concentration-dependent manner by making transient interactions with individual S100A9 molecules, perturbing its native structure and acting as catalysts. Since the individual molecule misfolding is a rate-limiting step in S100A9 amyloid aggregation, the effects of the NCAM1 construct on the native S100A9 are so critical for its amyloid self-assembly. S100A9 rapid self assembly into large aggregated clumps may prevent its amyloid tissue propagation, and by modulating S100A9 aggregation as a part of the amyloid cascade, the whole process may be effectively tuned.
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48.
  • Price, Neil P J, et al. (författare)
  • Branched Chain Lipid Metabolism As a Determinant of the N-Acyl Variation of Streptomyces Natural Products.
  • 2021
  • Ingår i: ACS chemical biology. - : American Chemical Society (ACS). - 1554-8937 .- 1554-8929. ; 16:1, s. 116-124
  • Tidskriftsartikel (refereegranskat)abstract
    • Branched-chain fatty acids (BCFA) are encountered in Gram-positive bacteria, but less so in other organisms. The bacterial BCFA in membranes are typically saturated, with both odd- and even-numbered carbon chain lengths, and with methyl branches at either the ω-1 (iso) or ω-2 (anteiso) positions. The acylation with BCFA also contributes to the structural diversity of microbial natural products and potentially modulates biological activity. For the tunicamycin (TUN) family of natural products, the toxicity toward eukaryotes is highly dependent upon N-acylation with trans-2,3-unsaturated BCFA. The loss of the 2,3-unsaturation gives modified TUN with reduced eukaryotic toxicity but crucially with retention of the synergistic enhancement of the β-lactam group of antibiotics. Here, we infer from genomics, mass spectrometry, and deuterium labeling that the trans-2,3-unsaturated TUN variants and the saturated cellular lipids found in TUN-producing Streptomyces are derived from the same pool of BCFA metabolites. Moreover, non-natural primers of BCFA metabolism are selectively incorporated into the cellular lipids of TUN-producing Streptomyces and concomitantly produce structurally novel neo-branched TUN N-acyl variants.
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49.
  • Rahman, Mahafuzur, et al. (författare)
  • Binding of Human Proteins to Amyloid-beta Protofibrils
  • 2015
  • Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 10:3, s. 766-774
  • Tidskriftsartikel (refereegranskat)abstract
    • The progressive neurodegeneration in Alzheimers disease is believed to be linked to the presence of prefibrillar aggregates of the amyloid-beta (A beta) peptide in the brain. The exact role of these aggregates in the disease pathology is, however, still an open question. Any mechanism by which oligomeric A beta may cause damage to neuronal cells must, in one way or another, involve interactions with other molecules. Here, we identify proteins in human serum and cerebrospinal fluid that bind to stable protofibrils formed by an engineered variant of A beta 42 (A beta(42CC)). We find that the protofibrils attract a substantial number of protein binding partners. Many of the 101 identified proteins are involved in lipid transport and metabolism, the complement system, or in hemostasis. Binding of representative proteins from all of these groups with micromolar affinity was confirmed using surface plasmon resonance. In addition, binding of apolipoprotein E to the protofibrils with nanomolar affinity was demonstrated. We also find that aggregation of A beta enhances protein binding, as lower amounts of proteins bind monomeric A beta. Proteins that bind to A beta protofibrils might contribute to biological effects in which these aggregates are involved. Our results therefore suggest that an improved understanding of the mechanisms by which A beta causes cytotoxicity and neurodegeneration might be gained from studies carried out in biologically relevant matrices in which A beta-binding proteins are present.
  •  
50.
  • Ranganathan, Anirudh, et al. (författare)
  • Ligand Discovery for a Peptide-Binding GPCR by Structure-Based Screening of Fragment- and Lead-Like Chemical Libraries
  • 2017
  • Ingår i: ACS Chemical Biology. - : AMER CHEMICAL SOC. - 1554-8929 .- 1554-8937. ; 12:3, s. 735-745
  • Tidskriftsartikel (refereegranskat)abstract
    • Peptide-recognizing G protein-coupled receptors (GPCRs) are promising therapeutic targets but often resist drug discovery efforts. Determination of crystal structures for peptide binding GPCRs has provided opportunities to explore structure based methods in lead development. Molecular docking screens of two chemical libraries, containing either fragment- or lead-like compounds, against a neurotensin receptor 1 crystal structure allowed for a comparison between different drug development strategies for peptide-binding GPCRs. A total of 2.3 million molecules were screened computationally, and 25 fragments and 27 leads that were top-ranked in each library were selected for experimental evaluation. Of these, eight fragments and five leads were confirmed as ligands by surface plasmon resonance. The hit rate for the fragment screen (32%) was thus higher than for the lead-like library (19%), but the affinities of the fragments were similar to 100-fold lower. Both screens returned unique scaffolds and demonstrated that a crystal structure of a stabilized peptide-binding GPCR can guide the discovery of small-molecule agonists. The complementary advantages of exploring fragment- and lead-like chemical space suggest that these strategies should be applied synergistically in structure-based screens against challenging GPCR targets.
  •  
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