36 results on '"Upert G"'
Search Results
2. Attribution of cystein's connectivities in conotoxins: new prospects based on partial oxidation/reduction experiments and ion-mobility mass spectrometry
- Author
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Quinton, L., Massonnet, P., Echterbille, J., Smargiasso, N., Upert, G, Pastor, A, Gilles, N, and De Pauw, E.
- Published
- 2014
3. 46. Efficient synthesis and refolding of two-disulfide-bridges toxins
- Author
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Upert, G., primary, Pastor, A., additional, Servent, D., additional, Mourier, G., additional, and Gilles, N., additional
- Published
- 2014
- Full Text
- View/download PDF
4. 8. Attribution of cystein’s connectivities in conotoxins: New prospects based on partial oxidation/reduction experiments and ion-mobility mass spectrometry
- Author
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Quinton, L., primary, Massonnet, P., additional, Echterbille, J., additional, Smargiasso, N., additional, Upert, G., additional, Pastor, A., additional, Gilles, N., additional, and De Pauw, E., additional
- Published
- 2014
- Full Text
- View/download PDF
5. VENOMICS Project: Peptidomics and transcriptomics of animal venoms for the discovery of novel therapeutic peptides
- Author
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Upert, G., primary, Pastor, A., additional, Servent, D., additional, Mourier, G., additional, and Gilles, N., additional
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- 2013
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- View/download PDF
6. An evolutionary approach of mamba venoms: “Le hasard et la nécessité”
- Author
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Blanchet, G., primary, Collet, G., additional, Gilles, N., additional, Mourier, G., additional, Upert, G., additional, and Servent, D., additional
- Published
- 2013
- Full Text
- View/download PDF
7. SOLUTION STRUCTURE OF THE SOMATOSTATIN SST3 RECEPTOR ANTAGONIST TAU- CONOTOXIN CnVA
- Author
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Petrel, C., primary, Hocking, H.G., additional, Reynaud, M., additional, Favreau, P., additional, Paolini-Bertrand, M., additional, Peigneur, S., additional, Upert, G., additional, Tytgat, J., additional, Gilles, N., additional, Hartley, O., additional, Boelens, R., additional, Stocklin, R., additional, and Servent, D., additional
- Published
- 2013
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- View/download PDF
8. Identification, structural and pharmacological characterization of τ-CnVA, a conopeptide that selectively interacts with somatostatin sst3 receptor.
- Author
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Petrel, C., Hocking, H.G., Reynaud, M., Upert, G., Favreau, Ph., Biass, D., Paolini-Bertrand, M., Peigneur, S., Tytgat, J., Gilles, N., Hartley, O., Boelens, R., Stocklin, R., and Servent, D.
- Subjects
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SOMATOSTATIN receptors , *MOLECULAR biology , *PEPTIDES , *ION channels , *DRUG synergism , *PHARMACOLOGY , *CONUS , *VENOM - Abstract
Abstract: Conopeptides are a diverse array of small linear and reticulated peptides that interact with high potency and selectivity with a large diversity of receptors and ion channels. They are used by cone snails for prey capture or defense. Recent advances in venom gland transcriptomic and venom peptidomic/proteomic technologies combined with bioactivity screening approaches lead to the identification of new toxins with original pharmacological profiles. Here, from transcriptomic/proteomic analyses of the Conus consors cone snail, we identified a new conopeptide called τ-CnVA, which displays the typical cysteine framework V of the T1-conotoxin superfamily. This peptide was chemically synthesized and its three-dimensional structure was solved by NMR analysis and compared to that of TxVA belonging to the same family, revealing very few common structural features apart a common orientation of the intercysteine loop. Because of the lack of a clear biological function associated with the T-conotoxin family, τ-CnVA was screened against more than fifty different ion channels and receptors, highlighting its capacity to interact selectively with the somatostatine sst3 receptor. Pharmacological and functional studies show that τ-CnVA displays a micromolar (Ki of 1.5μM) antagonist property for the sst3 receptor, being currently the only known toxin to interact with this GPCR subfamily. [Copyright &y& Elsevier]
- Published
- 2013
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9. Reply to: Antibiotics and hexagonal order in the bacterial outer membrane.
- Author
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Manioglu S, Modaresi SM, Thoma J, Overall SA, Upert G, Luther A, Barnes AB, Obrecht D, Müller DJ, and Hiller S
- Subjects
- Cell Membrane Permeability, Bacterial Outer Membrane Proteins metabolism, Anti-Bacterial Agents pharmacology, Bacterial Outer Membrane metabolism
- Published
- 2023
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10. Peptidomimetic antibiotics disrupt the lipopolysaccharide transport bridge of drug-resistant Enterobacteriaceae.
- Author
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Schuster M, Brabet E, Oi KK, Desjonquères N, Moehle K, Le Poupon K, Hell S, Gable S, Rithié V, Dillinger S, Zbinden P, Luther A, Li C, Stiegeler S, D'Arco C, Locher H, Remus T, DiMaio S, Motta P, Wach A, Jung F, Upert G, Obrecht D, Benghezal M, and Zerbe O
- Subjects
- Enterobacteriaceae, Lipopolysaccharides, Escherichia coli, Anti-Bacterial Agents pharmacology, Carrier Proteins, Peptidomimetics pharmacology, Escherichia coli Proteins
- Abstract
The rise of antimicrobial resistance poses a substantial threat to our health system, and, hence, development of drugs against novel targets is urgently needed. The natural peptide thanatin kills Gram-negative bacteria by targeting proteins of the lipopolysaccharide transport (Lpt) machinery. Using the thanatin scaffold together with phenotypic medicinal chemistry, structural data, and a target-focused approach, we developed antimicrobial peptides with drug-like properties. They exhibit potent activity against Enterobacteriaceae both in vitro and in vivo while eliciting low frequencies of resistance. We show that the peptides bind LptA of both wild-type and thanatin-resistant Escherichia coli and Klebsiella pneumoniae strains with low-nanomolar affinities. Mode of action studies revealed that the antimicrobial activity involves the specific disruption of the Lpt periplasmic protein bridge.
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- 2023
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11. Antibiotic polymyxin arranges lipopolysaccharide into crystalline structures to solidify the bacterial membrane.
- Author
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Manioglu S, Modaresi SM, Ritzmann N, Thoma J, Overall SA, Harms A, Upert G, Luther A, Barnes AB, Obrecht D, Müller DJ, and Hiller S
- Subjects
- Humans, Anti-Bacterial Agents pharmacology, Lipopolysaccharides, Escherichia coli, Polymyxin B pharmacology, Polymyxins pharmacology, Escherichia coli Infections
- Abstract
Polymyxins are last-resort antibiotics with potent activity against multi-drug resistant pathogens. They interact with lipopolysaccharide (LPS) in bacterial membranes, but mechanistic details at the molecular level remain unclear. Here, we characterize the interaction of polymyxins with native, LPS-containing outer membrane patches of Escherichia coli by high-resolution atomic force microscopy imaging, along with structural and biochemical assays. We find that polymyxins arrange LPS into hexagonal assemblies to form crystalline structures. Formation of the crystalline structures is correlated with the antibiotic activity, and absent in polymyxin-resistant strains. Crystal lattice parameters alter with variations of the LPS and polymyxin molecules. Quantitative measurements show that the crystalline structures decrease membrane thickness and increase membrane area as well as stiffness. Together, these findings suggest the formation of rigid LPS-polymyxin crystals and subsequent membrane disruption as the mechanism of polymyxin action and provide a benchmark for optimization and de novo design of LPS-targeting antimicrobials., (© 2022. The Author(s).)
- Published
- 2022
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12. From a Cone Snail Toxin to a Competitive MC4R Antagonist.
- Author
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Reynaud S, Laurin SA, Ciolek J, Barbe P, Van Baelen AC, Susset M, Blondel F, Ghazarian M, Boeri J, Vanden Driessche M, Upert G, Mourier G, Kessler P, Konnert L, Beroud R, Keck M, Servent D, Bouvier M, and Gilles N
- Subjects
- Amino Acid Sequence, Animals, Ligands, Melanocortins, Snails metabolism, Conotoxins pharmacology, Receptor, Melanocortin, Type 4
- Abstract
The melanocortin 4 receptor (MC4R) plays a role in energy homeostasis and represents a target for treating energy balance disorders. For decades, synthetic ligands have been derived from MC4R endogenous agonists and antagonists, such as setmelanotide used to treat rare forms of genetic obesity. Recently, animal venoms have demonstrated their capacity to provide melanocortin ligands with toxins from a scorpion and a spider. Here, we described a cone snail toxin, N-CTX-Ltg1a, with a nanomolar affinity for hMC4R but unrelated to any known toxins or melanocortin ligands. We then derived from the conotoxin the linear peptide HT1-0, a competitive antagonist of G
s , G15 , and β-arrestin2 pathways with a low nanomolar affinity for hMC4R. Similar to endogenous ligands, HT1-0 needs hydrophobic and basic residues to bind hMC4R. Altogether, it represents the first venom-derived peptide of high affinity on MC4R and paves the way for the development of new MC4R antagonists.- Published
- 2022
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13. A new Kunitz-type snake toxin family associated with an original mode of interaction with the vasopressin 2 receptor.
- Author
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Droctové L, Ciolek J, Mendre C, Chorfa A, Huerta P, Carvalho C, Gouin C, Lancien M, Stanajic-Petrovic G, Braco L, Blanchet G, Upert G, De Pauw G, Barbe P, Keck M, Mourier G, Mouillac B, Denis S, Rodríguez de la Vega RC, Quinton L, and Gilles N
- Subjects
- Animals, Peptides pharmacology, Rats, Snake Venoms pharmacology, Vasopressins, Elapidae metabolism, Receptors, Vasopressin metabolism
- Abstract
Background and Purpose: Venomous animals express numerous Kunitz-type peptides. The mambaquaretin-1 (MQ1) peptide identified from the Dendroaspis angusticeps venom is the most selective antagonist of the arginine-vasopressin V2 receptor (V2R) and the only unique Kunitz-type peptide active on a GPCR. We aimed to exploit other mamba venoms to enlarge the V2R-Kunitz peptide family and gain insight into the MQ1 molecular mode of action., Experimental Approach: We used a bio-guided screening assay to identify novel MQs and placed them phylogenetically. MQs were produced by solid-phase peptide synthesis and characterized in vitro by binding and functional tests and in vivo by diuresis measurement in rats., Key Results: Eight additional MQs were identified with nanomolar affinities for the V2R, all antagonists. MQs form a new subgroup in the Kunitz family, close to the V2R non-active dendrotoxins and to two V2R-active cobra toxins. Sequence comparison between active and non-active V2R Kunitz peptides highlighted five positions, among which four are involved in V2R interaction and belong to the two large MQ1 loops. We finally determined that eight positions, part of these two loops, interact with the V2R. The variant MQ1-K39A showed a higher affinity for the hV2R, but not for the rat V2R., Conclusions and Implications: A new function and mode of action is associated with the Kunitz peptides. The number of MQ1 residues involved in V2R binding is large and may explain its absolute selectivity. MQ1-K39A represents the first step in the improvement of the MQ1 design from a medicinal perspective., (© 2022 The British Pharmacological Society.)
- Published
- 2022
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14. Emerging peptide antibiotics with therapeutic potential.
- Author
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Upert G, Luther A, Obrecht D, and Ermert P
- Abstract
This review covers some of the recent progress in the field of peptide antibiotics with a focus on compounds with novel or established mode of action and with demonstrated efficacy in animal infection models. Novel drug discovery approaches, linear and macrocyclic peptide antibiotics, lipopeptides like the polymyxins as well as peptides addressing targets located in the plasma membrane or in the outer membrane of bacterial cells are discussed., Competing Interests: Gregory Upert, Daniel Obrecht and Philipp Ermert are employees of Polyphor Ltd. Anatol Luther is an employee of Bachem AG., (© 2021 The Authors.)
- Published
- 2021
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15. A Venomics Approach Coupled to High-Throughput Toxin Production Strategies Identifies the First Venom-Derived Melanocortin Receptor Agonists.
- Author
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Reynaud S, Ciolek J, Degueldre M, Saez NJ, Sequeira AF, Duhoo Y, Brás JLA, Meudal H, Cabo Díez M, Fernández Pedrosa V, Verdenaud M, Boeri J, Pereira Ramos O, Ducancel F, Vanden Driessche M, Fourmy R, Violette A, Upert G, Mourier G, Beck-Sickinger AG, Mörl K, Landon C, Fontes CMGA, Miñambres Herráiz R, Rodríguez de la Vega RC, Peigneur S, Tytgat J, Quinton L, De Pauw E, Vincentelli R, Servent D, and Gilles N
- Subjects
- Amino Acid Sequence, Animals, HEK293 Cells, High-Throughput Screening Assays methods, Humans, Receptors, Melanocortin metabolism, Scorpion Venoms genetics, Scorpion Venoms isolation & purification, Scorpion Venoms metabolism, Proteomics methods, Receptors, Melanocortin agonists, Scorpion Venoms pharmacology
- Abstract
Animal venoms are rich in hundreds of toxins with extraordinary biological activities. Their exploitation is difficult due to their complexity and the small quantities of venom available from most venomous species. We developed a Venomics approach combining transcriptomic and proteomic characterization of 191 species and identified 20,206 venom toxin sequences. Two complementary production strategies based on solid-phase synthesis and recombinant expression in Escherichia coli generated a physical bank of 3597 toxins. Screened on hMC4R, this bank gave an incredible hit rate of 8%. Here, we focus on two novel toxins: N-TRTX-Preg1a, exhibiting an inhibitory cystine knot (ICK) motif, and N-BUTX-Ptr1a, a short scorpion-CSαβ structure. Neither N-TRTX-Preg1a nor N-BUTX-Ptr1a affects ion channels, the known targets of their toxin scaffolds, but binds to four melanocortin receptors with low micromolar affinities and activates the hMC1R/Gs pathway. Phylogenetically, these two toxins form new groups within their respective families and represent novel hMC1R agonists, structurally unrelated to the natural agonists.
- Published
- 2020
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16. Can IM-MS Collision Cross Sections of Biomolecules Be Rationalized Using Collision Cross-Section Trends of Polydisperse Synthetic Homopolymers?
- Author
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Haler JRN, Massonnet P, Far J, Upert G, Gilles N, Mourier G, Quinton L, and De Pauw E
- Abstract
In the past, we developed a method inferring physicochemical properties from ion mobility mass spectrometry (IM-MS) data from polydisperse synthetic homopolymers. We extend here the method to biomolecules that are generally monodisperse. Similarities in the IM-MS behavior were illustrated on proteins and peptides. This allows one to identify ionic species for which intramolecular interactions lead to specific structures.
- Published
- 2020
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17. Combination of Capillary Zone Electrophoresis-Mass Spectrometry, Ion Mobility-Mass Spectrometry, and Theoretical Calculations for Cysteine Connectivity Identification in Peptides Bearing Two Intramolecular Disulfide Bonds.
- Author
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Delvaux C, Massonnet P, Kune C, Haler JRN, Upert G, Mourier G, Gilles N, Quinton L, De Pauw E, and Far J
- Subjects
- Electrophoresis, Capillary, Ion Mobility Spectrometry, Mass Spectrometry, Software, Cysteine analysis, Disulfides chemistry, Peptides chemistry
- Abstract
Disulfide bonds between cysteine residues are commonly involved in the stability of numerous peptides and proteins and are crucial for providing biological activities. In such peptides, the appropriate cysteine connectivity ensures the proper conformation allowing an efficient binding to their molecular targets. Disulfide bond connectivity characterization is still challenging and is a critical issue in the analysis of structured peptides/proteins targeting pharmaceutical or pharmacological utilizations. This study describes the development of new and fast gas-phase and in-solution electrophoretic methods coupled to mass spectrometry to characterize the cysteine connectivity of disulfide bonds. For this purpose, disulfide isomers of three peptides bearing two intramolecular disulfide bonds but different cysteine connectivity have been investigated. Capillary zone electrophoresis and ion mobility both coupled to mass spectrometry were used to perform the separation in both aqueous and gas phases, respectively. The separation efficiency of each technique has been critically evaluated and compared. Finally, theoretical calculations were performed to support and explain the experimental data based on the predicted physicochemical properties of the different peptides.
- Published
- 2020
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18. Author Correction: Chimeric peptidomimetic antibiotics against Gram-negative bacteria.
- Author
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Luther A, Urfer M, Zahn M, Müller M, Wang SY, Mondal M, Vitale A, Hartmann JB, Sharpe T, Monte FL, Kocherla H, Cline E, Pessi G, Rath P, Modaresi SM, Chiquet P, Stiegeler S, Verbree C, Remus T, Schmitt M, Kolopp C, Westwood MA, Desjonquères N, Brabet E, Hell S, LePoupon K, Vermeulen A, Jaisson R, Rithié V, Upert G, Lederer A, Zbinden P, Wach A, Moehle K, Zerbe K, Locher HH, Bernardini F, Dale GE, Eberl L, Wollscheid B, Hiller S, Robinson JA, and Obrecht D
- Abstract
An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
- Published
- 2019
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19. Chimeric peptidomimetic antibiotics against Gram-negative bacteria.
- Author
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Luther A, Urfer M, Zahn M, Müller M, Wang SY, Mondal M, Vitale A, Hartmann JB, Sharpe T, Monte FL, Kocherla H, Cline E, Pessi G, Rath P, Modaresi SM, Chiquet P, Stiegeler S, Verbree C, Remus T, Schmitt M, Kolopp C, Westwood MA, Desjonquères N, Brabet E, Hell S, LePoupon K, Vermeulen A, Jaisson R, Rithié V, Upert G, Lederer A, Zbinden P, Wach A, Moehle K, Zerbe K, Locher HH, Bernardini F, Dale GE, Eberl L, Wollscheid B, Hiller S, Robinson JA, and Obrecht D
- Subjects
- Animals, Anti-Bacterial Agents adverse effects, Bacterial Outer Membrane Proteins chemistry, Bacterial Outer Membrane Proteins genetics, Biological Products chemistry, Drug Discovery, Escherichia coli Proteins chemistry, Escherichia coli Proteins genetics, Fluorescence, Gram-Negative Bacteria genetics, Gram-Negative Bacteria pathogenicity, Humans, Lipopolysaccharides chemistry, Macrocyclic Compounds adverse effects, Macrocyclic Compounds chemistry, Macrocyclic Compounds pharmacology, Male, Mice, Microbial Sensitivity Tests, Microbial Viability drug effects, Microscopy, Electron, Transmission, Models, Molecular, Mutation, Peptidomimetics adverse effects, Photoaffinity Labels, Anti-Bacterial Agents chemistry, Anti-Bacterial Agents pharmacology, Drug Resistance, Microbial drug effects, Gram-Negative Bacteria drug effects, Peptidomimetics chemistry, Peptidomimetics pharmacology
- Abstract
There is an urgent need for new antibiotics against Gram-negative pathogens that are resistant to carbapenem and third-generation cephalosporins, against which antibiotics of last resort have lost most of their efficacy. Here we describe a class of synthetic antibiotics inspired by scaffolds derived from natural products. These chimeric antibiotics contain a β-hairpin peptide macrocycle linked to the macrocycle found in the polymyxin and colistin family of natural products. They are bactericidal and have a mechanism of action that involves binding to both lipopolysaccharide and the main component (BamA) of the β-barrel folding complex (BAM) that is required for the folding and insertion of β-barrel proteins into the outer membrane of Gram-negative bacteria. Extensively optimized derivatives show potent activity against multidrug-resistant pathogens, including all of the Gram-negative members of the ESKAPE pathogens
1 . These derivatives also show favourable drug properties and overcome colistin resistance, both in vitro and in vivo. The lead candidate is currently in preclinical toxicology studies that-if successful-will allow progress into clinical studies that have the potential to address life-threatening infections by the Gram-negative pathogens, and thus to resolve a considerable unmet medical need.- Published
- 2019
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20. Disulfide Connectivity Analysis of Peptides Bearing Two Intramolecular Disulfide Bonds Using MALDI In-Source Decay.
- Author
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Massonnet P, Haler JRN, Upert G, Smargiasso N, Mourier G, Gilles N, Quinton L, and De Pauw E
- Subjects
- Amino Acid Sequence, Conotoxins chemistry, Cysteine analysis, Isomerism, Oxidation-Reduction, Tandem Mass Spectrometry methods, Disulfides analysis, Peptides chemistry, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods
- Abstract
Disulfide connectivity in peptides bearing at least two intramolecular disulfide bonds is highly important for the structure and the biological activity of the peptides. In that context, analytical strategies allowing a characterization of the cysteine pairing are of prime interest for chemists, biochemists, and biologists. For that purpose, this study evaluates the potential of MALDI in-source decay (ISD) for characterizing cysteine pairs through the systematic analysis of identical peptides bearing two disulfide bonds, but not the same cysteine connectivity. Three different matrices have been tested in positive and/or in negative mode (1,5-DAN, 2-AB and 2-AA). As MALDI-ISD is known to partially reduce disulfide bonds, the data analysis of this study rests firstly on the deconvolution of the isotope pattern of the parent ions. Moreover, data analysis is also based on the formed fragment ions and their signal intensities. Results from MS/MS-experiments (MALDI-ISD-MS/MS) constitute the last reference for data interpretation. Owing to the combined use of different ISD-promoting matrices, cysteine connectivity identification could be performed on the considered peptides. Graphical Abstract ᅟ.
- Published
- 2018
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21. RgIA4 Potently Blocks Mouse α9α10 nAChRs and Provides Long Lasting Protection against Oxaliplatin-Induced Cold Allodynia.
- Author
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Christensen SB, Hone AJ, Roux I, Kniazeff J, Pin JP, Upert G, Servent D, Glowatzki E, and McIntosh JM
- Abstract
Transcripts for α9 and α10 nicotinic acetylcholine receptor (nAChR) subunits are found in diverse tissues. The function of α9α10 nAChRs is best known in mechanosensory cochlear hair cells, but elsewhere their roles are less well-understood. α9α10 nAChRs have been implicated as analgesic targets and α-conotoxins that block α9α10 nAChRs produce analgesia. However, some of these peptides show large potency differences between species. Additionally several studies have indicated that these conotoxins may also activate GABA
B receptors (GABAB Rs). To further address these issues, we cloned the cDNAs of mouse α9 and α10 nAChR subunits. When heterologously expressed in Xenopus oocytes, the resulting α9α10 nAChRs had the expected pharmacology of being activated by acetylcholine and choline but not by nicotine. A conotoxin analog, RgIA4, potently, and selectively blocked mouse α9α10 nAChRs with low nanomolar affinity indicating that RgIA4 may be effectively used to study murine α9α10 nAChR function. Previous reports indicated that RgIA4 attenuates chemotherapy-induced cold allodynia. Here we demonstrate that RgIA4 analgesic effects following oxaliplatin treatment are sustained for 21 days after last RgIA4 administration indicating that RgIA4 may provide enduring protection against nerve damage. RgIA4 lacks activity at GABAB receptors; a bioluminescence resonance energy transfer assay was used to demonstrate that two other analgesic α-conotoxins, Vc1.1 and AuIB, also do not activate GABAB Rs expressed in HEK cells. Together these findings further support the targeting of α9α10 nAChRs in the treatment of pain.- Published
- 2017
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22. Ancestral protein resurrection and engineering opportunities of the mamba aminergic toxins.
- Author
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Blanchet G, Alili D, Protte A, Upert G, Gilles N, Tepshi L, Stura EA, Mourier G, and Servent D
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- Amino Acid Sequence, Animals, Dendroaspis genetics, Evolution, Molecular, Models, Molecular, Phylogeny, Protein Conformation, Snake Venoms genetics, Snake Venoms pharmacology, Dendroaspis metabolism, Protein Engineering, Snake Venoms chemistry, Snake Venoms metabolism
- Abstract
Mamba venoms contain a multiplicity of three-finger fold aminergic toxins known to interact with various α-adrenergic, muscarinic and dopaminergic receptors with different pharmacological profiles. In order to generate novel functions on this structural scaffold and to avoid the daunting task of producing and screening an overwhelming number of variants generated by a classical protein engineering strategy, we accepted the challenge of resurrecting ancestral proteins, likely to have possessed functional properties. This innovative approach that exploits molecular evolution models to efficiently guide protein engineering, has allowed us to generate a small library of six ancestral toxin (AncTx) variants and associate their pharmacological profiles to key functional substitutions. Among these variants, we identified AncTx1 as the most α
1A -adrenoceptor selective peptide known to date and AncTx5 as the most potent inhibitor of the three α2 adrenoceptor subtypes. Three positions in the ρ-Da1a evolutionary pathway, positions 28, 38 and 43 have been identified as key modulators of the affinities for the α1 and α2C adrenoceptor subtypes. Here, we present a first attempt at rational engineering of the aminergic toxins, revealing an epistasis phenomenon.- Published
- 2017
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23. Ion Mobility-Mass Spectrometry as a Tool for the Structural Characterization of Peptides Bearing Intramolecular Disulfide Bond(s).
- Author
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Massonnet P, Haler JR, Upert G, Degueldre M, Morsa D, Smargiasso N, Mourier G, Gilles N, Quinton L, and De Pauw E
- Subjects
- Amino Acid Sequence, Cysteine, Protons, Disulfides analysis, Mass Spectrometry methods, Peptides chemistry
- Abstract
Disulfide bonds are post-translationnal modifications that can be crucial for the stability and the biological activities of natural peptides. Considering the importance of these disulfide bond-containing peptides, the development of new techniques in order to characterize these modifications is of great interest. For this purpose, collision cross cections (CCS) of a large data set of 118 peptides (displaying various sequences) bearing zero, one, two, or three disulfide bond(s) have been measured in this study at different charge states using ion mobility-mass spectrometry. From an experimental point of view, CCS differences (ΔCCS) between peptides bearing various numbers of disulfide bonds and peptides having no disulfide bonds have been calculated. The ΔCCS calculations have also been applied to peptides bearing two disulfide bonds but different cysteine connectivities (Cys1-Cys2/Cys3-Cys4; Cys1-Cys3/Cys2-Cys4; Cys1-Cys4/Cys2-Cys3). The effect of the replacement of a proton by a potassium adduct on a peptidic structure has also been investigated. Graphical Abstract ᅟ.
- Published
- 2016
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24. Acidosis increases MHC class II-restricted presentation of a protein endowed with a pH-dependent heparan sulfate-binding ability.
- Author
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Knittel D, Savatier A, Upert G, Lortat-Jacob H, and Léonetti M
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- Animals, Antigens genetics, Heparan Sulfate Proteoglycans genetics, Histocompatibility Antigens Class II genetics, Hydrogen-Ion Concentration, Mice, Mice, Inbred BALB C, Protein Binding, Antigen Presentation, Antigens immunology, Heparan Sulfate Proteoglycans immunology, Histocompatibility Antigens Class II immunology, T-Lymphocytes, Helper-Inducer immunology
- Abstract
Heparan sulfate proteoglycans (HSPGs) are ubiquitously expressed molecules that participate in numerous biological processes. We previously showed that HSPGs expressed on the surface of APCs can serve as receptors for a hybrid protein containing an HS ligand and an Ag, which leads to more efficient stimulation of Th cells. To investigate whether such behavior is shared by proteins with inherent HS-binding ability, we looked for proteins endowed with this characteristic. We found that diphtheria toxin and its nontoxic mutant, called CRM197, can interact with HS. However, we observed that their binding ability is higher at pH 6 than at pH 7.4. Therefore, as extracellular acidosis occurs during infection by various micro-organisms, we assessed whether HS-binding capacity affects MHC class II-restricted presentation at different pHs. We first observed that pH decrease allows CRM197 binding to HSPG-expressing cells, including APCs. Then, we showed that this interaction enhances Ag uptake and presentation to Th cells. Lastly, we observed that pH decrease does not affect processing and presentation abilities of the APCs. Our findings show that acidic pH causes an HSPG-mediated uptake and an enhancement of T cell stimulation of Ags with the inherent ability to bind HSPGs pH-dependently. Furthermore, they suggest that proteins from micro-organisms with this binding characteristic might be supported more efficiently by the adaptive immune system when acidosis is triggered during infection., (Copyright © 2015 by The American Association of Immunologists, Inc.)
- Published
- 2015
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25. Combined use of ion mobility and collision-induced dissociation to investigate the opening of disulfide bridges by electron-transfer dissociation in peptides bearing two disulfide bonds.
- Author
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Massonnet P, Upert G, Smargiasso N, Gilles N, Quinton L, and De Pauw E
- Subjects
- Amino Acid Sequence, Cysteine chemistry, Drug Design, Electron Transport, Molecular Sequence Data, Peptides chemical synthesis, Tandem Mass Spectrometry, Chemistry Techniques, Analytical methods, Disulfides chemistry, Peptides chemistry
- Abstract
Disulfide bonds are post-translational modifications (PTMs) often found in peptides and proteins. They increase their stability toward enzymatic degradations and provide the structure and (consequently) the activity of such folded proteins. The characterization of disulfide patterns, i.e., the cysteine connectivity, is crucial to achieve a global picture of the active conformation of the protein of interest. Electron-transfer dissociation (ETD) constitutes a valuable tool to cleave the disulfide bonds in the gas phase, avoiding chemical reduction/alkylation in solution. To characterize the cysteine pairing, the present work proposes (i) to reduce by ETD one of the two disulfide bridges of model peptides, resulting in the opening of the cyclic structures, (ii) to separate the generated species by ion mobility, and (iii) to characterize the species using collision-induced dissociation (CID). Results of this strategy applied to several peptides show different behaviors depending on the connectivity. The loss of SH· radical species, observed for all the peptides, confirms the cleavage of the disulfides during the ETD process.
- Published
- 2015
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26. High-throughput production of two disulphide-bridge toxins.
- Author
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Upert G, Mourier G, Pastor A, Verdenaud M, Alili D, Servent D, and Gilles N
- Subjects
- Amino Acid Sequence, Chromatography, High Pressure Liquid, Circular Dichroism, Molecular Sequence Data, Toxins, Biological chemistry, Toxins, Biological classification, Disulfides chemistry, Toxins, Biological metabolism
- Abstract
A quick and efficient production method compatible with high-throughput screening was developed using 36 toxins belonging to four different families of two disulphide-bridge toxins. Final toxins were characterized using HPLC co-elution, CD and pharmacological studies.
- Published
- 2014
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27. Zinc-selective inhibition of the promiscuous bacterial amide-hydrolase DapE: implications of metal heterogeneity for evolution and antibiotic drug design.
- Author
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Uda NR, Upert G, Angelici G, Nicolet S, Schmidt T, Schwede T, and Creus M
- Subjects
- Amidohydrolases chemistry, Amidohydrolases metabolism, Anti-Bacterial Agents chemistry, Anti-Bacterial Agents pharmacology, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Binding Sites, Biocatalysis drug effects, Captopril chemistry, Diaminopimelic Acid chemistry, Diaminopimelic Acid metabolism, Dose-Response Relationship, Drug, Drug Design, Drug Resistance, Bacterial drug effects, Isoenzymes antagonists & inhibitors, Isoenzymes chemistry, Isoenzymes metabolism, Kinetics, Manganese chemistry, Manganese pharmacology, Metalloproteins antagonists & inhibitors, Metalloproteins chemistry, Metalloproteins metabolism, Models, Molecular, Molecular Structure, Protein Structure, Tertiary, Salmonella enterica drug effects, Salmonella enterica enzymology, Salmonella enterica growth & development, Sulfhydryl Compounds chemistry, Sulfhydryl Compounds metabolism, Zinc pharmacology, Amidohydrolases antagonists & inhibitors, Bacterial Proteins antagonists & inhibitors, Captopril pharmacology, Zinc chemistry
- Abstract
The development of resistance to virtually all current antibiotics makes the discovery of new antimicrobial compounds with novel protein targets an urgent challenge. The dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) is an essential metallo-enzyme for growth and proliferation in many bacteria, acting in the desuccinylation of N-succinyl-L,L-diaminopimelic acid (SDAP) in a late stage of the anabolic pathway towards both lysine and a crucial building block of the peptidoglycan cell wall. L-Captopril, which has been shown to exhibit very promising inhibitory activity in vitro against DapE and has attractive drug-like properties, nevertheless does not target DapE in bacteria effectively. Here we show that L-captopril targets only the Zn(2+)-metallo-isoform of the enzyme, whereas the Mn(2+)-enzyme, which is also a physiologically relevant isoform in bacteria, is not inhibited. Our finding provides a rationale for the failure of this promising lead-compound to exhibit any significant antibiotic activity in bacteria and underlines the importance of addressing metallo-isoform heterogeneity in future drug design. Moreover, to our knowledge, this is the first example of metallo-isoform heterogeneity in vivo that provides an evolutionary advantage to bacteria upon drug-challenge.
- Published
- 2014
- Full Text
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28. Peptidomic comparison and characterization of the major components of the venom of the giant ant Dinoponera quadriceps collected in four different areas of Brazil.
- Author
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Cologna CT, Cardoso Jdos S, Jourdan E, Degueldre M, Upert G, Gilles N, Uetanabaro AP, Costa Neto EM, Thonart P, de Pauw E, and Quinton L
- Subjects
- Animals, Ants, Brazil, Ant Venoms chemistry, Insect Proteins chemistry, Peptides chemistry
- Abstract
Despite the noxious effects inflicted by Dinoponera ant's envenomation, the information about the biological properties and composition of their venom is still very limited. Ants from the genus Dinoponera are believed to be the world's largest living ants with a body length of 3cm. Their occurrence is restricted to tropical areas of South America. In this work, we study the venom of the giant Dinoponera quadriceps ant collected in 4 different regions of Brazil. By using a combination of complementary mass spectrometric approaches, we aim at: (i) characterizing the venom composition of these ants; (ii) establishing a comparative analysis of the venom from four geographically different regions in Brazil. This approach demonstrates that ant venom is a copious source of new compounds. Several peptides were identified and selected for "de novo sequencing". Since most of the new peptides showed similarities with antimicrobial peptides (AMPs), antimicrobial assays were performed with the purpose of evaluating their activity. In regard to the comparative study of the four regions, we observed not only major differences in the venom compositions, but also that the venoms collected in closest areas are more similar than the ones collected in distant regions. These observations seem to highlight an adaption of the ant venoms to the local environment. Concerning the biological assays, the peptides called Dq-3162 and Da-3177 showed a wide-ranging antimicrobial activity. The characterization of new AMPs with a broad spectrum of activity and different scaffolds may aid scientists to design new therapeutic agents and understand the mechanisms of those peptides to interact with microbial membranes. The results obtained betoken the biotechnological potential of ant's venom., Biological Significance: For the first time this manuscript describes an extensive proteomics characterization of the D. quadriceps venom. In addition this study reports the variation in venom composition of primitive ants from 4 geographically different areas of Brazil. The results reveal the presence of ~335 compounds for each venom/area and inter-colony variations were observed. 16 new peptides were characterized and 2 of them were synthesized and biologically assayed. These findings highlight the considerable and still unexplored diversity of ant's venom which could be used as valuable research tools in different areas of knowledge., (© 2013.)
- Published
- 2013
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29. New α-adrenergic property for synthetic MTβ and CM-3 three-finger fold toxins from black mamba.
- Author
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Blanchet G, Upert G, Mourier G, Gilquin B, Gilles N, and Servent D
- Subjects
- Adrenergic Agents chemical synthesis, Adrenergic Agents isolation & purification, Amino Acid Sequence, Animals, Circular Dichroism, Molecular Sequence Data, Protein Conformation, Receptors, Adrenergic metabolism, Receptors, Muscarinic metabolism, Snake Venoms chemical synthesis, Toxins, Biological chemical synthesis, Toxins, Biological isolation & purification, Adrenergic Agents chemistry, Elapidae, Snake Venoms chemistry, Toxins, Biological chemistry
- Abstract
Despite their isolation more than fifteen years ago from the venom of the African mamba Dendroaspis polylepis, very few data are known on the functional activity of MTβ and CM-3 toxins. MTβ was initially classified as a muscarinic toxin interacting non-selectively and with low affinity with the five muscarinic receptor subtypes while no biological function was determined for CM-3. Recent results highlight the multifunctional activity of three-finger fold toxins for muscarinic and adrenergic receptors and reveal some discrepancies in the pharmacological profiles of their venom-purified and synthetic forms. Here, we report the pharmacological characterization of chemically-synthesized MTβ and CM-3 toxins on nine subtypes of muscarinic and adrenergic receptors and demonstrate their high potency for α-adrenoceptors and in particular a sub-nanomolar affinity for the α1A-subtype. Strikingly, no or very weak affinity were found for muscarinic receptors, highlighting that pharmacological characterizations of venom-purified peptides may be risky due to possible contaminations. The biological profile of these two homologous toxins looks like that one previously reported for the Dendroaspis angusticeps ρ-Da1a toxin. Nevertheless, MTβ and CM-3 interact more potently than ρ-Da1a with α1B- and α1D-AR subtypes. A computational analysis of the stability of the MTβ structure suggests that mutation S38I, could be involved in this gain in function., (Copyright © 2013 Elsevier Ltd. All rights reserved.)
- Published
- 2013
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- View/download PDF
30. Oligoprolines as scaffolds for the formation of silver nanoparticles in defined sizes: correlating molecular and nanoscopic dimensions.
- Author
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Upert G, Bouillère F, and Wennemers H
- Subjects
- Aldehydes, Carboxylic Acids, Models, Molecular, Particle Size, Proline chemistry, Metal Nanoparticles chemistry, Oligopeptides chemistry, Silver chemistry
- Published
- 2012
- Full Text
- View/download PDF
31. Inhibition of HIV Replication by Cyclic and Hairpin PNAs Targeting the HIV-1 TAR RNA Loop.
- Author
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Upert G, Di Giorgio A, Upadhyay A, Manvar D, Pandey N, Pandey VN, and Patino N
- Abstract
Human immunodeficiency virus-1 (HIV-1) replication and gene expression entails specific interaction of the viral protein Tat with its transactivation responsive element (TAR), to form a highly stable stem-bulge-loop structure. Previously, we described triphenylphosphonium (TPP) cation-based vectors that efficiently deliver nucleotide analogs (PNAs) into the cytoplasm of cells. In particular, we showed that the TPP conjugate of a linear 16-mer PNA targeting the apical stem-loop region of TAR impedes Tat-mediated transactivation of the HIV-1 LTR in vitro and also in cell culture systems. In this communication, we conjugated TPP to cyclic and hairpin PNAs targeting the loop region of HIV-1 TAR and evaluated their antiviral efficacy in a cell culture system. We found that TPP-cyclic PNAs containing only 8 residues, showed higher antiviral potency compared to hairpin PNAs of 12 or 16 residues. We further noted that the TPP-conjugates of the 8-mer cyclic PNA as well as the 16-mer linear PNA displayed similar antiviral efficacy. However, cyclic PNAs were shown to be highly specific to their target sequences. This communication emphasizes on the importance of small constrained cyclic PNAs over both linear and hairpin structures for targeting biologically relevant RNA hairpins.
- Published
- 2012
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- View/download PDF
32. Nanoliter plates--versatile tools for the screening of split-and-mix libraries on-bead and off-bead.
- Author
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Upert G, Merten CA, and Wennemers H
- Subjects
- Alkaline Phosphatase antagonists & inhibitors, Alkaline Phosphatase metabolism, Combinatorial Chemistry Techniques, Dimethylpolysiloxanes chemistry, Enzyme Inhibitors chemical synthesis, Enzyme Inhibitors chemistry, Fluorescent Dyes chemistry, Small Molecule Libraries, beta-Galactosidase antagonists & inhibitors, beta-Galactosidase metabolism, Nanotubes chemistry
- Abstract
Nanoliter plates are versatile tools for the screening of split-and-mix libraries both on-bead and off-bead. The method was evaluated for the identification of hydrolytically active compounds and enzyme inhibitors.
- Published
- 2010
- Full Text
- View/download PDF
33. Enamine catalysis with low catalyst loadings--high efficiency via kinetic studies.
- Author
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Wiesner M, Upert G, Angelici G, and Wennemers H
- Abstract
Kinetic studies on enamine catalysis provided insight into the rate determining step(s) of peptide catalyzed conjugate addition reactions between aldehydes and nitroolefins. They demonstrate that not enamine formation but both the reaction of the enamine with the electrophile and hydrolysis of the resulting imine are rate limiting. These results allowed for reducing the catalyst loading by a factor of 10 to as little as 0.1 mol %. This is the lowest catalyst loading that has been achieved so far in enamine catalysis with low molecular weight catalysts for a broad range of substrates.
- Published
- 2010
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- View/download PDF
34. Silver nanoparticle formation in different sizes induced by peptides identified within split-and-mix libraries.
- Author
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Belser K, Vig Slenters T, Pfumbidzai C, Upert G, Mirolo L, Fromm KM, and Wennemers H
- Subjects
- Peptides chemistry, Combinatorial Chemistry Techniques, Metal Nanoparticles chemistry, Peptide Library, Peptides isolation & purification, Silver chemistry
- Abstract
Split-and-mix libraries are an excellent tool for the identification of peptides that induce the formation of Ag nanoparticles in the presence of either light or sodium ascorbate to reduce Ag(+) ions. Structurally diverse peptides were detected in colorimetric on-bead screenings that generate Ag nanoparticles of different sizes, as confirmed by SEM and X-ray powder diffraction studies.
- Published
- 2009
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35. An efficient biodelivery system for antisense polyamide nucleic acid (PNA).
- Author
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Mehiri M, Upert G, Tripathi S, Di Giorgio A, Condom R, Pandey VN, and Patino N
- Subjects
- Anti-HIV Agents metabolism, Anti-HIV Agents pharmacology, Cell Line, Culture Media, HIV Long Terminal Repeat drug effects, HIV-1 drug effects, HIV-1 genetics, HIV-1 physiology, Humans, Nylons chemistry, Peptide Nucleic Acids chemical synthesis, Peptide Nucleic Acids pharmacology, Virus Replication drug effects, Antisense Elements (Genetics), Drug Delivery Systems, Nylons metabolism, Peptide Nucleic Acids metabolism
- Abstract
With the aim of developing a general and straightforward procedure for the intracellular delivery of naked peptide nucleic acids (PNAs), we designed an intracellularly biodegradable triphenylphosphonium (TPP) cation based transporter system. In this system, TPP is linked, via a biolabile disulfide bridge, to an activated mercaptoethoxycarbonyl moiety, allowing its direct coupling to the N-terminal extremity of a free PNA through a carbamate bond. We found that such TPP-PNA-carbamate conjugates were highly stable in a cell culture medium containing fetal calf serum. In a glutathione-containing medium mimicking the cytosol, the conjugates were rapidly degraded into an unstable intermediate, which spontaneously decomposed, releasing the free PNA. Using a fluorescence-labeled PNA-TPP conjugate, we demonstrated that conjugates were taken up by cells. Efficient cellular uptake and release of the PNA into the cytosol was further confirmed by the anti-HIV activity measured for the TPP-conjugate of a 16-mer PNA targeting the TAR region of the HIV-1 genome. This conjugate exhibited an IC(50) value of 1 microM, while the free 16-mer PNA did not inhibit replication of HIV in the same cellular test.
- Published
- 2008
- Full Text
- View/download PDF
36. Solid-phase synthesis and thermal denaturation study of cyclic PNAs targeting the HIV-1 TAR RNA loop.
- Author
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Upert G, Mehiri M, Di Giorgio A, Condom R, and Patino N
- Subjects
- Peptide Nucleic Acids chemistry, Peptide Nucleic Acids pharmacology, Peptides, Cyclic chemistry, Peptides, Cyclic pharmacology, Spectrophotometry, Ultraviolet, HIV-1 genetics, Peptide Nucleic Acids chemical synthesis, Peptides, Cyclic chemical synthesis, RNA, Viral drug effects
- Abstract
Cyclic PNAs targeting the HIV-1 TAR RNA loop have been synthesized following a convenient solid-phase strategy which allows on-resin cyclisation. UV-monitored thermal denaturation studies demonstrate that these cyclic PNAs are able to strongly interact with their TAR RNA target, very likely through the formation of a six-base pair stable complex, involving the TAR RNA loop.
- Published
- 2007
- Full Text
- View/download PDF
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