| 1. |
- Altincekic, Nadide, et al.
(författare)
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Targeting the Main Protease (Mpro, nsp5) by Growth of Fragment Scaffolds Exploiting Structure-Based Methodologies
- 2024
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Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 19:2, s. 563-574
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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. |
- Andersson, Hanna, 1979, et al.
(författare)
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Assessing the Ability of Spectroscopic Methods to Determine the Difference in the Folding Propensities of Highly Similar beta-Hairpins
- 2017
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Ingår i: Acs Omega. - : American Chemical Society (ACS). - 2470-1343. ; 2:2, s. 508-516
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Tidskriftsartikel (refereegranskat)abstract
- We have evaluated the ability of nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopies to describe the difference in the folding propensities of two structurally highly similar cyclic beta-hairpins, comparing the outcome to that of molecular dynamics simulations. NAMFIS-type NMR ensemble analysis and CD spectroscopy were observed to accurately describe the consequence of altering a single interaction site, whereas a single-site C-13 NMR chemical shift melting curve-based technique was not.
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| 3. |
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| 4. |
- Andersson, Hanna, 1979, et al.
(författare)
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Solvent Effects on Nitrogen Chemical Shifts
- 2015
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Ingår i: Annual Reports on NMR Spectroscopy. - : Elsevier. - 0066-4103. - 9780128021231 ; 86, s. 73-210
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Forskningsöversikt (refereegranskat)abstract
- Due to significant developments in cryogenic probe technology and the easy access to inverse detection pulse programmes (HSQC, HMBC), the sensitivity of nitrogen NMR has lately vastly improved. As a consequence, nitrogen NMR has turned into a useful and commonly available tool for solution studies of molecular structure and properties for small organic compounds likewise biopolymers. The high sensitivity of the nitrogen lone pair to changes in the molecular environment, alterations in intra- and intermolecular interactions, and in molecular conformation along with its wide, up to 1200 ppm chemical shift dispersion make nitrogen NMR to an exceptionally sensitive reporter tool. The nitrogen chemical shift has been applied in various fields of chemistry, including for instance the studies of transition metal complexes, chemical reactions such as N-alkylation and N-oxidation, tautomerization, protonation–deprotonation equilibria, hydrogen and halogen bonding, and elucidation of molecular conformation and configuration. The 15N NMR data observed in the investigation of these molecular properties and processes is influenced by the medium it is acquired in. This influence may be due to direct coordination of solvent molecules to transition metal complexes, alteration of tautomerization equilibria, and solvent polarity induced electron density changes of conjugated systems, for example. Thus, the solvent may significantly alter the observed nitrogen NMR shifts. This review aims to provide an overview of solvent effects of practical importance, and discusses selected experimental reports from various subfields of chemistry.
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| 5. |
- Blobel, Jascha, et al.
(författare)
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Protein loop compaction and the origin of the effect of arginine and glutamic acid mixtures on solubility, stability and transient oligomerization of proteins
- 2011
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Ingår i: European Biophysics Journal. - : Springer Science and Business Media LLC. - 0175-7571 .- 1432-1017. ; 40:12, s. 1327-1338
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Tidskriftsartikel (refereegranskat)abstract
- Addition of a 50 mM mixture of l-arginine and l-glutamic acid (RE) is extensively used to improve protein solubility and stability, although the origin of the effect is not well understood. We present Small Angle X-ray Scattering (SAXS) and Nuclear Magnetic Resonance (NMR) results showing that RE induces protein compaction by collapsing flexible loops on the protein core. This is suggested to be a general mechanism preventing aggregation and improving resistance to proteases and to originate from the polyelectrolyte nature of RE. Molecular polyelectrolyte mixtures are expected to display long range correlation effects according to dressed interaction site theory. We hypothesize that perturbation of the RE solution by dissolved proteins is proportional to the volume occupied by the protein. As a consequence, loop collapse, minimizing the effective protein volume, is favored in the presence of RE.
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| 6. |
- Brath, Ulrika, et al.
(författare)
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Differential responses of the backbone and side-chain conformational dynamics in FKBP12 upon binding the transition-state analog FK506: implications for transition-state stabilization and target protein recognition.
- 2009
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 387:1, s. 233-244
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Tidskriftsartikel (refereegranskat)abstract
- FKBP12 serves a dual role as a peptidyl-prolyl cis-trans isomerase and as a modulator of several cell signaling pathways. The macrolide FK506 is a transition-state analog of the catalyzed reaction and displaces FKBP12 from its natural target proteins. We compared the conformational exchange dynamics of the backbone and methyl-bearing side chains of FKBP12 in the free and FK506-bound states using NMR relaxation-dispersion experiments. Our results show that the free enzyme exchanges between the ground state and an excited state that resembles the ligand-bound state or Michaelis complex. In FK506-bound FKBP12, the backbone is confined to a single conformation, while conformational exchange prevails for many methyl groups. The residual side-chain dynamics in the transition-state analog-bound state suggests that the transition-state ensemble involves multiple conformations, a finding that challenges the long-standing concept of conformational restriction in the transition-state complex. Furthermore, exchange between alternative conformations is observed in the bound state for an extended network of methyl groups that includes locations remote from the active site. Several of these locations are known to be important for interactions with cellular target proteins, including calcineurin and the ryanodine receptor, suggesting that the conformational heterogeneity might play a role in the promiscuous binding of FKBP12 to different targets.
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| 7. |
- Brath, Ulrika, et al.
(författare)
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Functional dynamics of human FKBP12 revealed by methyl C-13 rotating frame relaxation dispersion NMR spectroscopy
- 2006
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 128:17, s. 5718-5727
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Tidskriftsartikel (refereegranskat)abstract
- Transverse relaxation dispersion NMR spectroscopy can provide atom-specific information about time scales, populations, and the extent of structural reorganization in proteins under equilibrium conditions. A method is described that uses side-chain methyl groups as local reporters for conformational transitions taking place in the microsecond regime. The experiment measures carbon nuclear spin relaxation rates in the presence of continuous wave off-resonance irradiation, in proteins uniformly enriched with C-13, and partially randomly labeled with 2 H. The method was applied to human FK-506 binding protein (FKBP12), which uses a common surface for binding substrates in its dual role as both an immunophilin and folding assistant. Conformational dynamics on a time scale of similar to 130 mu s were detected for methyl groups located in the substrate binding pocket, demonstrating its plasticity in the absence of substrate. The spatial arrangement of affected side-chain atoms suggests that substrate recognition involves the rapid relative movement of the subdomain comprising residues Ala81-Thr96 and that the observed dynamics play an important role in facilitating the interaction of this protein with its many partners, including calcineurin.
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| 8. |
- Brath, Ulrika, et al.
(författare)
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Mapping the sevoflurane-binding site of calmodulin
- 2014
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Ingår i: Pharmacology Research & Perspectives. - : Wiley. - 2052-1707 .- 2052-1707. ; 2:1
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Tidskriftsartikel (refereegranskat)abstract
- General anesthetics, with sevoflurane (SF) being the first choice inhalational anesthetic agent, provide reversible, broad depressor effects on the nervous system yet have a narrow margin of safety. As characterization of low-affinity binding interactions of volatile substances is exceptionally challenging with the existing methods, none of the numerous cellular targets proposed as chief protagonists in anesthesia could yet be confirmed. The recognition that most critical functions modulated by volatile anesthetics are under the control of intracellular Ca2+ concentration, which in turn is primarily regulated by calmodulin (CaM), motivated us for characterization of the SF–CaM interaction. Solution NMR (Nuclear Magnetic Resonance) spectroscopy was used to identify SF-binding sites using chemical shift displacement, NOESY and heteronuclear Overhauser enhancement spectroscopy (HOESY) experiments. Binding affinities were measured using ITC (isothermal titration calorimetry). SF binds to both lobes of (Ca2+)4-CaM with low mmol/L affinity whereas no interaction was observed in the absence of Ca2+. SF does not affect the calcium binding of CaM. The structurally closely related SF and isoflurane are shown to bind to the same clefts. The SF-binding clefts overlap with the binding sites of physiologically relevant ion channels and bioactive small molecules, but the binding affinity suggests it could only interfere with very weak CaM targets.
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| 9. |
- Brath, Ulrika
(författare)
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Molecular dynamics studied by NMR relaxation experiments. Characterization of functional dynamics in the FK506 binding protein FKBP12.
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The presented thesis work is concerned with the study of molecular dynamics using liquid nuclear magnetic resonance (NMR) spectroscopy. Spin relaxation dispersion experiments in the rotating frame (R1ρ) are employed to provide a detailed, atomic-resolution view of protein dynamics. Comprehensive investigations of 15N backbone and 13C methyl side-chain conformational exchange in the FK506 binding protein, FKBP12, were carried out. The human enzyme FKBP12 catalyzes the cis-trans isomerization of peptidyl-prolyl linkages in proteins, thereby increasing the rate of protein folding. Recombinant protein production with 13C/2H-enrichment yielded 13CHD2 isotopomers and allowed for development of novel R1ρ experiments aimed to characterize the methyl-bearing side-chains. The joint analysis of backbone and side-chain dynamics revealed a collective conformational exchange process with a correlation time of 121 ± 26 microseconds in the free enzyme. The locations of the exchanging nuclei showed close correspondence with residues previously shown to be important for the catalytic activity, which emphasize the connection between protein function and dynamics. The FKBP12 isomerase activity is strongly inhibited by the small organic molecule FK506. In addition, the bound conformation of FK506 exhibit distinct structural transition state analog characteristics. Conformational exchange dynamics studies of the FKBP12 – FK506 complex demonstrated a striking rigidity of the backbone atoms, in line with the established theory that the transition state is more constrained than the free state. Conformational exchange dynamics found for peripheral methyl groups is suggested to play a role in the subsequent binding of the FKBP12 – FK506 complex to other target molecules. A specific isotope labeling strategy using [1-13C]-glucose was shown to produce isolated 13C nuclei in the side-chains of Phe/Tyr/Trp/His. Proteins with this characteristic labeling provide nuclei amenable to R1ρ experiments, which were previously excluded from analysis due to complications arising from uniform carbon labeling. The method thus extended the collection of dynamic probes to include the frequently occurring and often functionally important aromatic side-chains. R1ρ studies of the C-terminal part of a mutant Calmodulin protein revealed microsecond time scale dynamics contributions to Tyr80Cδ, confirming previous reports of conformational exchange present for the protein backbone nuclei. Detection of conformational exchange in isotropic media requires a chemical shift difference between the exchanging states. However, such chemical shift difference can be replaced by differential residual dipolar couplings (RDC) in the exchanging states for molecules dissolved in partially aligning media. The RDC-induced contribution to the exchange-free transverse relaxation rate can be suppressed using relaxation dispersion experiments, thus revealing details of the conformational exchange characteristics. The theory was described, and the method was demonstrated using the model molecule N,N-dimethyltrichloroacetamide.
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| 10. |
- Brath, Ulrika, et al.
(författare)
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Paramagnetic ligand tagging to identify protein binding sites
- 2015
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 137:35, s. 11391-11398
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Tidskriftsartikel (refereegranskat)abstract
- Transient biomolecular interactions are the cornerstones of the cellular machinery. The identification of the binding sites for low affinity molecular encounters is essential for the development of high affinity pharmaceuticals from weakly binding leads but is hindered by the lack of robust methodologies for characterization of weakly binding complexes. We introduce a paramagnetic ligand tagging approach that enables localization of low affinity protein–ligand binding clefts by detection and analysis of intermolecular protein NMR pseudocontact shifts, which are invoked by the covalent attachment of a paramagnetic lanthanoid chelating tag to the ligand of interest. The methodology is corroborated by identification of the low millimolar volatile anesthetic interaction site of the calcium sensor protein calmodulin. It presents an efficient route to binding site localization for low affinity complexes and is applicable to rapid screening of protein–ligand systems with varying binding affinity.
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