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  • 1.
    Elinder, Malin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Nordström, Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Geitmann, Matthis
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Hämäläinen, Markku
    Vrang, Lotta
    Öberg, Bo
    Danielson, U Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Screening for NNRTIs with Slow Dissociation and High Affinity for a Panel of HIV-1 RT Variants2009In: Journal of Biomolecular Screening, ISSN 1087-0571, E-ISSN 1552-454X, Vol. 14, no 4, p. 395-403Article in journal (Refereed)
    Abstract [en]

    A lead optimization library consisting of 800 HIV-1 nonnucleoside reverse transcriptase inhibitors (NNRTIs) was screened in parallel against 4 clinically relevant variants of HIV-1 RT (Wt, L100I, Y181C, and K103N) using a surface plasmon resonance-based biosensor. the aim was to identify inhibitors suitable in specific topical microbicides efficient for preventing the transmission of a range of clinically significant strains of HIV-1. the authors hypothesized that such compounds should have high affinity and slow dissociation rates for multiple variants of the target. to efficiently analyze the large amount of real-time data (sensorgrams) that were generated in the  screening, they initially used signals from 3 selected time points to identify compounds with high affinity and slow dissociation for the   complete panel of enzyme variants. hits were confirmed by visually  inspecting the complete sensorgrams. two structurally unrelated   compounds fulfilled the hit criteria, but only 1 compound was found to   (a) compete with a known NNRTI for binding to the NNRTI site, (b)   inhibit HIV-1 RT activity, and (c) inhibit HIV-1 replication in cell culture, for all 4 enzyme variants. this novel screening methodology offers high-resolution real-time kinetic data for multiple targets in parallel. it is expected to have broad applicability for the discovery of compounds with defined kinetic profiles, crucial for optimal therapeutic effects.

  • 2.
    Geitmann, Matthis
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Dahl, Göran
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mechanistic and kinetic characterization of hepatitis C virus NS3 protein interactions with NS4A and protease inhibitors2011In: Journal of Molecular Recognition, ISSN 0952-3499, E-ISSN 1099-1352, Vol. 24, no 1, p. 60-70Article in journal (Refereed)
    Abstract [en]

    The mechanism and kinetics of the interactions between ligands and immobilized full-length hepatitis C virus (HCV) genotype 1a NS3 have been characterized by SPR biosensor technology. The NS3 interactions for a series of NS3 protease inhibitors as well as for the NS4A cofactor, represented by a peptide corresponding to the sequence interacting with the enzyme, were found to be heterogeneous. It may represent interactions with two stable conformations of the protein. The NS3-NS4A interaction consisted of a high-affinity (K(D) = 50 nM) and a low-affinity (K(D) = 2 µM) interaction, contributing equally to the overall binding. By immobilizing NS3 alone or together with NS4A it was shown that all inhibitors had a higher affinity for NS3 in the presence of NS4A. NS4A thus has a direct effect on the binding of inhibitors to NS3 and not only on catalysis. As predicted, the mechanism-based inhibitor VX 950 exhibited a time-dependent interaction with a slow formation of a stable complex. BILN 2061 or ITMN-191 showed no signs of time-dependent interactions, but ITMN-191 had the highest affinity of the tested compounds, with both the slowest dissociation (k(off)) and fastest association rate, closely followed by BILN 2061. The k(off) for the inhibitors correlated strongly with their NS3 protease inhibitory effect as well as with their effect on replication of viral proteins in replicon cell cultures, confirming the relevance of the kinetic data. This approach for obtaining kinetic and mechanistic data for NS3 protease inhibitor and cofactor interactions is expected to be of importance for understanding the characteristics of HCV NS3 functionality as well as for anti-HCV lead discovery and optimization.

  • 3.
    Geitmann, Matthis
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Additional level of information about complex interaction between non-nucleoside inhibitor and HIV-1 reverse transcriptase using biosensor-based thermodynamic analysis2007In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 15, no 23, p. 7344-7354Article in journal (Refereed)
    Abstract [en]

    The thermodynamics of the interaction between mutant HIV-1 reverse transcriptase (K103N and Y181C) and a nonnucleoside reverse transcriptase inhibitor (NNRTI), the phenylethylthiazolylurea compound MIV-150, was obtained by determining the temperature dependence of the kinetic rate constants. Large entropic changes in the forward and backward steps of the isomerization between a non-binding competent and a binding competent conformation of the enzyme, as well as in the binding steps, implied the involvement of major structural rearrangements upon interaction with the inhibitor. Despite of the entropic character of the overall interaction, the equilibrium for the binding of inhibitor was found to be predominantly enthalpy-driven. The high affinity and the low affinity interactions of the heterogeneously interacting inhibitor showed different energetics in the analysis, revealing an expectedly higher enthalpic component for the high-affinity interaction. The thermodynamic profiles of the two enzyme variants displayed significant differences, which could not be derived from their kinetics at a single temperature.

  • 4.
    Geitmann, Matthis
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Studies of substrate-induced conformational changes in human cytomegalovirus protease using optical biosensor technology2004In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 332, no 2, p. 203-214Article in journal (Refereed)
    Abstract [en]

    The interaction between human cytomegalovirus (HCMV) protease and a peptide substrate was studied using a surface plasmon resonance (SPR)-based biosensor. Immobilization of the enzyme to the sensor chip surface by amine coupling resulted in an active enzyme with a higher catalytic efficiency than the enzyme in solution, primarily due to a lower K(m) value. The interaction between immobilized protease and substrate was characterized by a biphasic SPR signal. Rate constants for the formation of the initial enzyme-substrate complex could be determined from the sensorgrams. Simulated binding curves based on the determined k(cat) and the rate constants indicated that the complex binding signal did not originate from the accumulation of intermediates in the catalytic reaction. By chemical crosslinking of the immobilized HCMV protease, which was shown to limit the enzyme's structural flexibility, it was revealed that the obtained sensorgrams were composed of a signal caused by substrate binding and considerable structural alterations in the immobilized enzyme. Furthermore, HCMV protease was inactivated by chemical crosslinking, indicating that structural flexibility is essential for this enzyme. Parallel experiments with immobilized alpha-chymotrypsin revealed that it does not undergo similar conformational changes on peptide binding and that crosslinking did not inactivate the enzyme. The simultaneous detection of binding and conformational changes using optical biosensor technology is expected to be of importance for further characterization of the enzymatic properties of HCMV protease and for identification of inhibitors of this enzyme. It can also be of use for studies of other flexible proteins.

  • 5.
    Norgren, Anna S.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Geitmann, Matthis
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Arvidsson, Per I.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Biomolecular Recognition of Glycosylated β3-Peptides by GalNAc Specific Lectins2007In: Journal of Molecular Recognition, ISSN 0952-3499, E-ISSN 1099-1352, Vol. 20, no 2, p. 132-138Article in journal (Refereed)
    Abstract [en]

    The molecular recognition of a novel kind of hybrid conjugates, composed of artificial biomimetic β-peptide oligomers with an O-linked natural N-acetyl-galactosamine (the Tn-antigen) residue, by four different GalNAc specific lectins was investigated using surface plasmon biosensor technology. The influence of the peptide and the glycosyl moiety on the recognition was studied using two glycosylated β3-heptapeptides, a glycosylated α-heptapeptide, two β-amino acid containing dipeptides, and monomeric αGalNAc-O-Thr. Although all four lectins displayed a decreased affinity for the carbohydrate residue when attached to a peptide, as compared to the monomeric Tn-antigen, the peptide part was found to have distinct effects on the binding kinetics - indicating that varying degrees of protein-peptide interactions occurred in the recognition process. Likewise, the lectins did not discriminate between β3-peptides and the α-peptide, but the β- linkage of the galactose had a detrimental effect for at least two of the lectins.

  • 6. Simister, Philip
    et al.
    Schmitt, Melanie
    Geitmann, Matthis
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Wicht, Oliver
    Danielson, Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Klein, Rahel
    Bressanelli, Stephane
    Lohmann, Volker
    Structural and Functional Analysis of Hepatitis C Virus Strain JFH1 Polymerase2009In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 83, no 22, p. 11926-11939Article in journal (Refereed)
    Abstract [en]

    The hepatitis C virus (HCV) isolate JFH1 represents the only cloned wild-type sequence capable of efficient replication in cell culture, as well as in chimpanzees. Previous reports have pointed to the viral polymerase NS5B as a major determinant for efficient replication of this isolate. To understand the underlying mechanisms, we expressed and purified NS5B of JFH1 and of the closely related isolate J6, which replicates below the limit of detection in cell culture. The JFH1 enzyme exhibited a 5- to 10-fold-higher specific activity in vitro, consistent with the polymerase activity itself contributing to efficient replication of JFH1. The higher in vitro activity of the JFH1 enzyme was not due to increased RNA binding, elongation rate, or processivity of the polymerase but to higher initiation efficiency. By using homopolymeric and heteropolymeric templates, we found that purified JFH1 NS5B was significantly more efficient in de novo initiation of RNA synthesis than the J6 counterpart, particularly at low GTP concentrations, probably representing an important prerequisite for the rapid replication kinetics of JFH1. Furthermore, we solved the crystal structure of JFH1 NS5B, which displays a very closed conformation that is expected to facilitate de novo initiation. Structural analysis shows that this closed conformation is stabilized by a sprinkle of substitutions that together promote extra hydrophobic interactions between the subdomains "thumb" and "fingers." These analyses provide deeper insights into the initiation of HCV RNA synthesis and might help to establish more efficient cell culture models for HCV using alternative isolates.

  • 7. Wang, Xiaolu
    et al.
    Hu, Bin
    Zieba, Agata
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Neumann, Nicole G
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Kasper-Sonnenberg, Monika
    Honsbein, Annegret
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Conze, Tim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Witt, Wolfgang
    Limbach, Christoph
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Geitmann, Matthis
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Danielson, Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Kolarow, Richard
    Niemann, Gesa
    Lessmann, Volkmar
    Kilimann, Manfred W
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    A protein interaction node at the neurotransmitter release site: domains of Aczonin/Piccolo, Bassoon, CAST, and rim converge on the N-terminal domain of Munc13-12009In: Journal of Neuroscience, ISSN 0270-6474, E-ISSN 1529-2401, Vol. 29, no 40, p. 12584-12596Article in journal (Refereed)
    Abstract [en]

    Multidomain scaffolding proteins organize the molecular machinery of neurotransmitter vesicle dynamics during synaptogenesis and synaptic activity. We find that domains of five active zone proteins converge on an interaction node that centers on the N-terminal region of Munc13-1 and includes the zinc-finger domain of Rim1, the C-terminal region of Bassoon, a segment of CAST1/ELKS2, and the third coiled-coil domain (CC3) of either Aczonin/Piccolo or Bassoon. This multidomain complex may constitute a center for the physical and functional integration of the protein machinery at the active zone. An additional connection between Aczonin and Bassoon is mediated by the second coiled-coil domain of Aczonin. Recombinant Aczonin-CC3, expressed in cultured neurons as a green fluorescent protein fusion protein, is targeted to synapses and suppresses vesicle turnover, suggesting involvements in synaptic assembly as well as activity. Our findings show that Aczonin, Bassoon, CAST1, Munc13, and Rim are closely and multiply interconnected, they indicate that Aczonin-CC3 can actively participate in neurotransmitter vesicle dynamics, and they highlight the N-terminal region of Munc13-1 as a hub of protein interactions by adding three new binding partners to its mechanistic potential in the control of synaptic vesicle priming.

1 - 7 of 7
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