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  • 1.
    Abdurakhmanov, Eldar
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Solbak, Sara Oie
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Biophysical Mode-of-Action and Selectivity Analysis of Allosteric Inhibitors of Hepatitis C Virus (HCV) Polymerase2017In: Viruses, ISSN 1999-4915, E-ISSN 1999-4915, Vol. 9, no 6, 151Article in journal (Refereed)
    Abstract [en]

    Allosteric inhibitors of hepatitis C virus (HCV) non-structural protein 5B (NS5B) polymerase are effective for treatment of genotype 1, although their mode of action and potential to inhibit other isolates and genotypes are not well established. We have used biophysical techniques and a novel biosensor-based real-time polymerase assay to investigate the mode-of-action and selectivity of four inhibitors against enzyme from genotypes 1b (BK and Con1) and 3a. Two thumb inhibitors (lomibuvir and filibuvir) interacted with all three NS5B variants, although the affinities for the 3a enzyme were low. Of the two tested palm inhibitors (dasabuvir and nesbuvir), only dasabuvir interacted with the 1b variant, and nesbuvir interacted with NS5B 3a. Lomibuvir, filibuvir and dasabuvir stabilized the structure of the two 1b variants, but not the 3a enzyme. The thumb compounds interfered with the interaction between the enzyme and RNA and blocked the transition from initiation to elongation. The two allosteric inhibitor types have different inhibition mechanisms. Sequence and structure analysis revealed differences in the binding sites for 1b and 3a variants, explaining the poor effect against genotype 3a NS5B. The indirect mode-of-action needs to be considered when designing allosteric compounds. The current approach provides an efficient strategy for identifying and optimizing allosteric inhibitors targeting HCV genotype 3a.

  • 2.
    Ahlsén, Göran
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Hultén, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Shuman, Cynthia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Poliakov, Anton
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Lindgren, Maria T.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Alterman, Mathias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Samuelsson, Bertil
    Hallberg, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Resistance profiles of cyclic and linear inhibitors of HIV-1 protease2002In: Antiviral Chemistry & Chemotherapy, ISSN 0956-3202, E-ISSN 2040-2066, Vol. 13, no 1, 27-37 p.Article in journal (Refereed)
    Abstract [en]

    Resistance to anti-HIV protease drugs is a major problem in the design of AIDS drugs with long-term efficacy. To identify structural features associated with a certain resistance profile, the inhibitory properties of a series of symmetric and asymmetric cyclic sulfamide, cyclic urea and linear transition-state analogue inhibitors of HIV-1 protease were investigated using wild-type and mutant enzyme. To allow a detailed structure-inhibition analysis, enzyme with single, double, triple and quadruple combinations of G48V, V82A, 184V and L90M substitutions was used. Kinetic analysis of the mutants revealed that catalytic efficiency was 1-30% of that for the wild-type enzyme, a consequence of reduced kcat in all cases and an increased KM for all mutants except for the G48V enzyme. The overall structure-inhibitory profiles of the cyclic compounds were similar, and the inhibition of the V82A, 184V and G48V/L90M mutants were less efficient than of the wild-type enzyme. The greatest increase in Ki was generally observed for the 184V mutant and least for the G48V/L90M mutant, and additional combinations of mutations did not result in improved inhibition profiles for the cyclic compounds. An extended analysis of additional mutants, and including a set of linear compounds, showed that the profile was unique for each compound, and did not reveal any general structural features associated with a certain inhibition profile. The effects of structural modifications in the inhibitors, or of mutations, were not additive and they differed depending on their context. The results demonstrate the difficulties in predicting resistance, even for closely related compounds, and designing compounds with improved resistance profiles.

  • 3.
    Alterman, Mathias
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Andersson, Hans O.
    Garg, Neeraj
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Ahlsén, Göran
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Lövgren, Seved
    Classon, Björn
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Kvarnström, Ingemar
    Vrang, Lotta
    Unge, Torsten
    Samuelsson, Bertil
    Hallberg, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Design and fast synthesis of C-terminal duplicated potent C2-symmetric P1/P1'-modified HIV-1 protease inhibitors1999In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 42, no 19, 3835-3844 p.Article in journal (Refereed)
  • 4.
    Alterman, Mathias
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Björsne, Magnus
    Mühlman, Anna
    Classon, Björn
    Kvarnstrom, Ingemar
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Markgren, Per-Olof
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Nillroth, Ulrika
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Unge, Torsten
    Hallberg, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Samuelsson, Bertil
    Design and synthesis of new potent C2-symmetric HIV-1 protease inhibitors: Use of L-mannaric acid as a peptidomimetic scaffold1998In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 41, no 20, 3782-3792 p.Article in journal (Refereed)
  • 5.
    Alterman, Mathias
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Sjöbom, Hans
    Säfsten, Pär
    Markgren, Per-Olof
    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.
    Hämäläinen, Markku
    Löfås, Stefan
    Hultén, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Classon, Björn
    Samuelsson, Bertil
    Hallberg, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    P1/P1' modified HIV protease inhibitors as tools in two new sensitive surface plasmon resonance biosensor screening assays2001In: European Journal of Pharmaceutical Sciences, ISSN 0928-0987, E-ISSN 1879-0720, Vol. 13, no 2, 203-212 p.Article in journal (Refereed)
    Abstract [en]

    The commonly used HIV-1 protease assays rely on measurements of the effect of inhibitions on the hydrolysis rate of synthetic peptides. Recently an assay based on surface plasmon resonance (SPR) was introduced. We have taken advantage of the fact that the SPR signal is proportional to the mass of the analyte interacting with the immobilised molecule and developed two new improved efficient competition assay methods. Thus, high molecular weight binders were used as amplifiers of the surface plasmon resonance signal. Linkers were attached by a Heck reaction to the para-positions of the P1/P1′ benzyloxy groups of a linear C2-symmetric C-terminal duplicated inhibitor to enable (a) biotin labelling or (b) direct immobilisation of the inhibitor to the biosensor surface matrix. The interaction properties of a series of 17 structurally diverse inhibitors was assessed and compared to previously reported data. The most sensitive assay was obtained by immobilising the enzyme and amplifying the signal with an antibody, giving a detection range between 0.1 nM and 10 μM. Immobilisation of the inhibitor resulted in a stable and durable surface but a narrower detection range (1–100 nM). The two competition assays are anticipated to be very suitable for fast screening of potential HIV inhibitors.

  • 6.
    Andersson, Hans O.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Fridborg, Kerstin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Löwgren, Seved
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Alterman, Mathias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Mühlman, Anna
    Björsne, Magnus
    Garg, Neeraj
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Kvarnström, Ingemar
    Schaal, Wesley
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Classon, Björn
    Karlén, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Ahlsén, Göran
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Nillroth, Ulrika
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Vrang, Lotta
    Öberg, Bo
    Samuelsson, Bertil
    Hallberg, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Unge, Torsten
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Optimization of P1-P3 groups in symmetric and asymmetric HIV-1 protease inhibitors2003In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 270, no 8, 1746-1758 p.Article in journal (Refereed)
  • 7.
    Anscombe, Elizabeth
    et al.
    Univ Oxford, Dept Biochem, Oxford OX1 3QU, England..
    Meschini, Elisa
    Newcastle Univ, Sch Chem, Northern Inst Canc Res, Newcastle Canc Ctr, Newcastle Upon Tyne NE1 7RU, Tyne & Wear, England..
    Mora-Vidal, Regina
    Newcastle Univ, Newcastle Canc Ctr, Northern Inst Canc Res, Sch Med, Newcastle Upon Tyne NE2 4HH, Tyne & Wear, England..
    Martin, Mathew P.
    Newcastle Univ, Newcastle Canc Ctr, Northern Inst Canc Res, Sch Med, Newcastle Upon Tyne NE2 4HH, Tyne & Wear, England..
    Staunton, David
    Univ Oxford, Dept Biochem, Oxford OX1 3QU, England..
    Geitmann, Matthis
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Stanley, Will A.
    Newcastle Univ, Newcastle Canc Ctr, Northern Inst Canc Res, Sch Med, Newcastle Upon Tyne NE2 4HH, Tyne & Wear, England..
    Wang, Lan Z.
    Newcastle Univ, Newcastle Canc Ctr, Northern Inst Canc Res, Sch Med, Newcastle Upon Tyne NE2 4HH, Tyne & Wear, England..
    Reuillon, Tristan
    Newcastle Univ, Sch Chem, Northern Inst Canc Res, Newcastle Canc Ctr, Newcastle Upon Tyne NE1 7RU, Tyne & Wear, England..
    Golding, Bernard T.
    Newcastle Univ, Sch Chem, Northern Inst Canc Res, Newcastle Canc Ctr, Newcastle Upon Tyne NE1 7RU, Tyne & Wear, England..
    Cano, Celine
    Newcastle Univ, Sch Chem, Northern Inst Canc Res, Newcastle Canc Ctr, Newcastle Upon Tyne NE1 7RU, Tyne & Wear, England..
    Newell, David R.
    Newcastle Univ, Newcastle Canc Ctr, Northern Inst Canc Res, Sch Med, Newcastle Upon Tyne NE2 4HH, Tyne & Wear, England..
    Noble, Martin E. M.
    Univ Oxford, Dept Biochem, Oxford OX1 3QU, England..
    Wedge, Stephen R.
    Newcastle Univ, Newcastle Canc Ctr, Northern Inst Canc Res, Sch Med, Newcastle Upon Tyne NE2 4HH, Tyne & Wear, England..
    Endicott, Jane A.
    Univ Oxford, Dept Biochem, Oxford OX1 3QU, England..
    Griffin, Roger J.
    Newcastle Univ, Sch Chem, Northern Inst Canc Res, Newcastle Canc Ctr, Newcastle Upon Tyne NE1 7RU, Tyne & Wear, England..
    Identification and Characterization of an Irreversible Inhibitor of CDK22015In: Chemistry and Biology, ISSN 1074-5521, E-ISSN 1879-1301, Vol. 22, no 9, 1159-1164 p.Article in journal (Refereed)
    Abstract [en]

    Irreversible inhibitors that modify cysteine or lysine residues within a protein kinase ATP binding site offer, through their distinctive mode of action, an alternative to ATP-competitive agents. 4-((6-(Cyclo-hexylmethoxy)-9H-purin-2-yl) amino) benzenesulfonamide (NU6102) is a potent and selective ATP-competitive inhibitor of CDK2 in which the sulfonamide moiety is positioned close to a pair of lysine residues. Guided by the CDK2/NU6102 structure, we designed 6-(cyclohexylmethoxy)-N-(4-(vinylsulfonyl) phenyl)-9H- purin-2-amine (NU6300), which binds covalently to CDK2 as shown by a co-complex crystal structure. Acute incubation with NU6300 produced a durable inhibition of Rb phosphorylation in SKUT-1B cells, consistent with it acting as an irreversible CDK2 inhibitor. NU6300 is the first covalent CDK2 inhibitor to be described, and illustrates the potential of vinyl sulfones for the design of more potent and selective compounds.

  • 8.
    Backman, Dan
    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.
    Kinetic and mechanistic analysis of the association and dissociation of inhibitors interacting with secreted aspartic acid proteases 1 and 2 from Candida albicans2003In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1646, no 1-2, 184-195 p.Article in journal (Refereed)
  • 9.
    Backman, Dan
    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.
    Substrate specificity and pH dependence of secreted aspartic proteases Sap1, Sap2 and Sap3 from Candida albicansManuscript (Other (popular science, discussion, etc.))
  • 10.
    Backman, Dan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Monod, Michel
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Biosensor-based screening and characterization of HIV-1 inhibitor interactions with Sap1, Sap2 and Sap3 from Candida albicans2006In: Journal of Biomolecular Screening, ISSN 1087-0571, E-ISSN 1552-454X, Vol. 11, no 2, 165-175 p.Article in journal (Refereed)
  • 11. Barreca, Maria Letizia
    et al.
    Manfroni, Giuseppe
    Leyssen, Pieter
    Winquist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Kaushik-Basu, Neerja
    Paeshuyse, Jan
    Krishnan, Ramalingam
    Iraci, Nunzio
    Sabatini, Stefano
    Tabarrini, Oriana
    Basu, Amartya
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Neyts, Johan
    Cecchetti, Violetta
    Structure-Based Discovery of Pyrazolobenzothiazine Derivatives As Inhibitors of Hepatitis C Virus Replication2013In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 56, no 6, 2270-2282 p.Article in journal (Refereed)
    Abstract [en]

    The NS5B RNA-dependent RNA polymerase is an attractive target for the development of novel and selective inhibitors of hepatitis C virus replication. To identify novel structural hits as anti-HCV agents, we performed structure based virtual screening of our in-house library followed by rational drug design, organic synthesis, and biological testing. These studies led to the identification of pyrazolobenzothiazine scaffold as a suitable template for obtaining novel anti-HCV agents targeting the NS5B polymerase. The best compound of this series was the meta-fluoro-N-1-phenyl pyrazolobenzothiazine derivative 4a, which exhibited an EC50 = 3.6 mu M, EC90 = 25.6 mu M, and CC50 > 180 mu M in the Huh 9-13 replicon system, thus providing a good starting point for further hit evolution.

  • 12.
    Belfrage, Anna Karin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Abdurakhmanov, Eldar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Åkerblom, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Brandt, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Oshalim, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Gising, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Skogh, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Neyts, Johan
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Sandström, Anja
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Discovery of pyrazinone based compounds that potently inhibit the drug resistant enzyme variant R155K of the hepatitis C virus NS3 protease2016In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 24, no 12, 2603-2620 p.Article in journal (Refereed)
    Abstract [en]

    Herein, we present the design and synthesis of 2(1H)-pyrazinone based HCV NS3 protease inhibitors with variations in the C-terminus. Biochemical evaluation was performed using genotype 1a, both the wildtype and the drug resistant enzyme variant, R155K. Surprisingly, compounds without an acidic sulfonamide retained good inhibition, challenging our previous molecular docking model. Moreover, selected compounds in this series showed nanomolar potency against R155K NS3 protease; which generally confer resistance to all HCV NS3 protease inhibitors approved or in clinical trials. These results further strengthen the potential of this novel substance class, being very different to the approved drugs and clinical candidates, in the development of inhibitors less sensitive to drug resistance.

  • 13. Belfrage, Anna-Karin
    et al.
    Gising, Johan
    Örtqvist, Pernilla
    Alogheli, Hiba
    Ehrenberg, Angelica
    Larhed, Mats
    Danielson, U Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Sandström, Anja
    Synthesis and SAR Around the R6 Position of 2(1H)-Pyrazinone Based HCV NS3 Protease Inhibitors2011In: Biopolymers, ISSN 0006-3525, E-ISSN 1097-0282, Vol. 96, 457-457 p.Article in journal (Refereed)
  • 14. Belfrage, Anna-Karin
    et al.
    Gising, Johan
    Örtqvist, Pernilla
    Danielson, U Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Larhed, Mats
    Sandström, Anja
    Hepatitis C Virus NS3 Protease Inhibitors based on 2(1H)-Pyrazinones2010In: Journal of Peptide Science, ISSN 1075-2617, E-ISSN 1099-1387, Vol. 16, 95-95 p.Article in journal (Refereed)
  • 15. Bertucci, Carlo
    et al.
    Pistolozzi, Marco
    Felix, Guy
    Danielson, Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    HSA binding of HIV protease inhibitors: a high-performance affinity chromatography study2009In: Journal of Separation Science, ISSN 1615-9306, E-ISSN 1615-9314, Vol. 32, no 10, 1625-1631 p.Article in journal (Refereed)
    Abstract [en]

    The binding of HIV protease inhibitors, drugs important for anti-HIV chemotherapy, to HSA was examined by high-performance affinity chromatography. Frontal analysis was first used to determine the amount of anchored protein and the binding capacity for selected markers on this column. Zonal elution experiments then ranked the HSA bound fraction of the examined compounds. Information on the g region was obtained by competitive zonal elution experiments using probe binding compounds with known sites on HSA. An allosteric competition between HIV protease inhibitors (PIs) and valproate (a probe for the bilirubin site) was detected, consistent with a noncooperative binding mechanism. No significant competition was observed between the examined compounds and salicylate or ibuprofen, probes for sites I and II, respectively. The observations were confirmed by circular dichroism spectroscopy, based on the change in the induced circular dichroism signals of selected markers for the main binding sites of HSA when ritonavir was added as the competitor. These results were in good agreement with previous literature reports and provide more details on how PIs are transported in plasma and how they may compete with other drugs in the body.

  • 16. Beyer, B
    et al.
    Rubin, J
    Johnson, J
    Chung, A
    Hayashi, Y
    Andersson, D
    Kiso, Y
    Danielson, Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Dunn, B
    Candida albicans secreted aspartic peptidase specificity: A chromogenic combinatorial approach2003In: Biopolymers, ISSN 0006-3525, E-ISSN 1097-0282, Vol. 71, no 3, 310-P025 p.Article in journal (Other academic)
  • 17. Blumenzweig, I
    et al.
    Baraz, L
    Friedler, A
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Gilon, C
    Steinitz, M
    Kotler, M
    HIV-1 Vif-derived peptide inhibits drug-resistant HIV proteases2002In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 292, no 4, 832-840 p.Article in journal (Refereed)
  • 18. Brandt, Peter
    et al.
    Geitmann, Matthis
    Danielson, U Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Deconstruction of Non-Nucleoside Reverse Transcriptase Inhibitors of Human Immunodeficiency Virus Type 1 for Exploration of the Optimization Landscape of Fragments2011In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804Article in journal (Refereed)
    Abstract [en]

    This study has taken a closer look at the theoretical basis for protein-fragment interactions. The approach involved the deconstruction of 3 non-nucleoside inhibitors of HIV-1 reverse transcriptase and investigation of the interaction between 21 substructures and the enzyme. It focused on the concept of ligand efficiency and showed that ligand independent free energy fees (ΔG(ind)) are crucial for the understanding of the binding affinities of fragments. A value of 7.0 kcal mol(-1) for the ΔG(ind) term is shown to be a lower limit for the NNRTI binding pocket of HIV-1 RT. The addition of the ΔG(ind) term to the dissociation free energy in the calculation of a corrected ligand efficiency, in combination with the lack of an efficient ligand binding hot spot in the NNIBP, fully explains the existence of nonbinding NNRTI substructures. By applying the concept to a larger set of ligands, we could define a binding site profile that indicates the absence of an efficient fragment binding hot spot but an efficient binding of full-sized NNRTIs. The analysis explains some of the challenges in identifying fragments against flexible targets involving conformational changes and how fragments may be prioritized.

  • 19. Brånalt, J
    et al.
    Kvarnstrom, I
    Classon, B
    Samuelsson, B
    Nillroth, Ulrika
    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.
    Karlén, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Hallberg, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    A convenient synthesis of 1-(S)-[1'-(S)-(t-butyloxycarbonylamino)-2'-phenylethyl]oxirane: a useful building block in the synthesis of HIV protease inhibitors1997In: Tetrahedron Letters, ISSN 0040-4039, E-ISSN 1359-8562, Vol. 38, no 19, 3483-3486 p.Article in journal (Refereed)
  • 20. Bäckbro, Kristina
    et al.
    Andersson, Hans
    Bonham, Nick M
    Classon, Björn
    Danielson, U Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Hallberg, Anders
    Hultén, Johan
    Karlén, Anders
    Löwgren, Seved
    Nillroth, Ulrika
    Schaal, Wesley
    Unge, Torsten
    Cyclic urea and sulfamide-based inhibitors of HIV-1 protease: Changes in binding mode upon modifications in the P1/P1 side chain1998In: Antiviral Research, ISSN 0166-3542, E-ISSN 1872-9096, Vol. 37, A56-A56 p.Article in journal (Refereed)
  • 21.
    Chaga, Grigoriy
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Andersson, Lennart
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Porath, Jerker
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Danielson, U Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Engineering of a metal coordinating site into human glutathione transferase M1-1 based on immobilized metal ion affinity chromatography of homologous rat enzymes1994In: Protein Engineering, ISSN 0269-2139, E-ISSN 1460-213X, Vol. 7, no 9, 1115-1119 p.Article in journal (Refereed)
    Abstract [en]

    Rat glutathione transferase (GST) 3-3 binds to Ni(II)-iminodiacetic acid (IDA)-agarose, whereas other GSTs that are abundant in rat liver do not bind to this immobilized metal ion affinity chromatography (IMAC) adsorbent. Rat GST 3-3 contains two superficially located amino acid residues, His84 and His85, that are suitably positioned for coordination to Ni(II)-IDA-agarose. This particular structural motif is lacking in GSTs that do not bind to the IMAC matrix. Creation of an equivalent His-His structure in the homologous human GST M1-1 by protein engineering afforded a mutant enzyme that displays affinity for Ni(II)-IDA-agarose, in contrast to the wild-type GST M1-1. The results identify a distinct site that is operational in IMAC and suggest an approach to the rational design of novel integral metal coordination sites in proteins.

  • 22. Christopeit, Tony
    et al.
    Gossas, Thomas
    Danielson, Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Characterization of Ca2+ and phosphocholine interactions with C-reactive protein using a surface plasmon resonance biosensor2009In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 391, no 1, 39-44 p.Article in journal (Refereed)
    Abstract [en]

    The interactions between Ca2+ and C-reactive protein (CRP) have been characterized using a surface plasmon resonance (SPR) biosensor. The protein was immobilized on a sensor chip, and increasing concentrations of Ca2+ or phosphocholine were injected. Binding of Ca2+ induced a 10-fold higher signal than expected from the molecular weight of Ca2+. It was interpreted to result from the conformational change that occurs on binding of Ca2+. Two sites with different characteristics were distinguished: a high-affinity site with K-D = 0.03 mM and a low-affinity site with K-D = 5.45 mM. The pH dependencies of the two Ca2+ interactions were different and enabled the assignment of the different sites in the three-dimensional structure of CRP. There was no evidence for cooperativity in the phosphocholine interaction, which had K-D = 5 mu M at 10 mM Ca2+. SPR biosensors can clearly detect and quantify the binding of very small molecules or ions to immobilized proteins despite the theoretically very low signals expected on binding, provided that significant conformational changes are involved. Both the interactions and the conformational changes can be characterized. The data have important implications for the understanding of the function of CRP and Suggest that Ca2+ is an efficient regulator under physiological conditions.

  • 23.
    Christopeit, Tony
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Stenberg, Gun
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Gossas, Thomas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Nyström, Susanne
    Baraznenok, Vera
    Lindström, Erik
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    A surface plasmon resonance-based biosensor with full-length BACE1 in a reconstituted membrane2011In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 414, no 1, 14-22 p.Article in journal (Refereed)
    Abstract [en]

    A surface plasmon resonance (SPR) biosensor-based assay for membrane-embedded full-length BACE1 (β-site amyloid precursor protein cleaving enzyme 1), a drug target for Alzheimer's disease, has been developed. It allows the analysis of interactions with the protein in its natural lipid membrane environment. The enzyme was captured via an antibody recognizing a C-terminal His6 tag, after which a lipid membrane was reconstituted on the chip using a brain lipid extract. The interaction between the enzyme and several inhibitors confirmed that the surface was functional. It had slightly different interaction characteristics as compared with a reference surface with immobilized ectodomain BACE1 but had the same inhibitor characteristic pH effect. The possibility of studying interactions with BACE1 under more physiological conditions than assays using truncated enzyme or conditions dictated by high enzyme activity is expected to increase our understanding of the role of BACE1 in Alzheimer's disease and contribute to the discovery of clinically efficient BACE1 inhibitors. The strategy exploited in the current study can be adapted to other membrane-bound drug targets by selecting suitable capture antibodies and lipid mixtures for membrane reconstitution.

  • 24.
    Christopeit, Tony
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Øverbø, Kersti
    Nofima AS, Tromsø, Norway.
    Danielson, Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Nilsen, Inge W.
    Nofima AS, Tromsø, Norway.
    Efficient Screening of Marine Extracts for Protease Inhibitors by Combining FRET Based Activity Assays and Surface Plasmon Resonance Spectroscopy Based Binding Assays2013In: Marine Drugs, ISSN 1660-3397, E-ISSN 1660-3397, Vol. 11, no 11, 4279-4293 p.Article in journal (Refereed)
    Abstract [en]

    The screening of extracts from marine organisms is a widely used strategy to discover new drug leads. A common problem in the screening process is the generation of false positive hits through unspecific effects from the complex chemical composition of the crude extracts. In this study, we explored a combination of a fluorescence resonance energy transfer (FRET) based activity assay and a surface plasmon resonance (SPR) based binding assay to avoid this problem. An aqueous extract was prepared from rest raw material of the Norwegian spring spawning herring, and further fractionated by methanol solubility and solid phase extraction. FRET based activity assays were used to determine the influence of each extract on the activity of different proteases. Several extracts showed more than 50% inhibition. The inhibition mechanisms were elucidated by SPR based competition experiments with known inhibitors. For the secreted aspartic proteases 1, 2, 3 and HIV-1 protease, the results indicated that some extracts contain inhibitors interacting specifically with the active site of the enzymes. The study shows that a combination of an activity assay and an SPR based binding assay is a powerful tool to identify potent inhibitors in marine extracts. Furthermore, the study shows that marine vertebrates offer an interesting source for new bioactive compounds, although they have rarely been explored for this purpose.

  • 25.
    Cimitan, Samanta
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Lindgren, Maria T.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Bertucci, Carlo
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Early absorption and distribution analysis of antitumor and anti-AIDS drugs: lipid membrane and plasma protein interactions2005In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 48, no 10, 3536-46 p.Article in journal (Refereed)
  • 26.
    Dahl, Göran
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Gutiérrez Arenas, Omar
    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.
    Hepatitis C Virus NS3 Protease Is Activated by Low Concentrations of  Protease Inhibitors2009In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 48, 11592-11602 p.Article in journal (Refereed)
    Abstract [en]

    The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) is a   bifunctional enzyme with a protease and a helicase functionality   located in each of the two domains of the single peptide chain. There   is little experimental evidence for a functional role of this   unexpected arrangement since artificial single domain forms of both   enzymes are catalytically competent. We have observed that low   concentrations of certain protease inhibitors activate the protease of   full-length NS3 from HCV genotype 1a with up to 100%, depending on the   preincubation time and the inhibitor used. The activation was reduced,   but not eliminated, by increased ionic strength, lowered glycerol   concentration, or lowered pH. In all cases, it was at the expense of a   significant loss of activity. Activation was not seen with the   artificial protease domain of genotype 1b NS3 fused with a fragment of   the NS4A cofactor. This truncated and covalently modified enzyme form   was much less active and exhibited fundamentally different catalytic   properties to the full-length NS3 protease without (he fused cofactor.   The most plausible explanation for the activation was found to involve   a slow transition between two enzyme conformations, which differed in   their catalytic ability and affinity for inhibitors. Equations derived   based on this assumption resulted in better fits to the experimental   data than the equation for simple competitive inhibition. The mechanism   may involve an inhibitor-induced stabilization of the helicase domain   in a conformation that enhances the protease activity, or all improved   alignment of the catalytic triad in the protease. The proposed mnemonic   mechanism and derived equations are viable for both these explanations   and can serve as a basic framework for future studies of enzymes   activated by inhibitors or other ligands.

  • 27.
    Dahl, Göran
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Sandström, Anja
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Åkerblom, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Effects on protease inhibition by modifying of helicase residues in hepatitis C virus nonstructural protein 32007In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 274, no 22, 5979-5986 p.Article in journal (Refereed)
    Abstract [en]

    This study of the full-length bifunctional nonstructural protein 3 from hepatitis C virus (HCV) has revealed that residues in the helicase domain affect the inhibition of the protease. Two residues (Q526 and H528), apparently located in the interface between the S2 and S4 binding pockets of the substrate binding site of the protease, were selected for modification, and three enzyme variants (Q526A, H528A and H528S) were expressed, purified and characterized. The substitutions resulted in indistinguishable Km values and slightly lower kcat values compared to the wild-type. The Ki values for a series of structurally diverse protease inhibitors were affected by the substitutions, with increases or decreases up to 10-fold. The inhibition profiles for H528A and H528S were different, confirming that not only did the removal of the imidazole side chain have an effect, but also that minor differences in the nature of the introduced side chain influenced the characteristics of the enzyme. These results indicate that residues in the helicase domain of nonstructural protein 3 can influence the protease, supporting our hypothesis that full-length hepatitis C virus nonstructural protein 3 should be used for protease inhibitor optimization and characterization. Furthermore, the data suggest that inhibitors can be designed to interact with residues in the helicase domain, potentially leading to more potent and selective compounds.

  • 28.
    Dahl, Göran
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Sandström, Anja
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Åkerblom, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Resistance profiling of hepatitis C virus protease inhibitors using full-length NS32007In: Antiviral Therapy, ISSN 1359-6535, E-ISSN 2040-2058, Vol. 12, no 5, 733-740 p.Article in journal (Refereed)
    Abstract [en]

    Background: The NS3 protease of hepatitis C virus (HCV) is a prime target for anti-HCV drugs but resistance towards inhibitors of the enzyme is likely to emerge because of mutations in the viral genome that modify the structure of the protein. Enzyme inhibition data supporting this is limited to studies with few compounds and analysis performed with truncated NS3.

    Experimental: The potential of HCV acquiring resistance towards NS3 protease inhibitors and the structural features associated with resistance has been explored with a series of inhibitors and by using full-length NS3 protease/helicase variants with amino acid substitutions (A156T, D168V and R155Q) in the protease domain.

    Results: The A156T and D168V substitutions did not influence the kinetic properties of the protease, whereas the R155Q substitution reduced the catalytic efficiency 20 times, as compared with the wild type. Inhibition studies revealed that these substitutions primarily affected the potency of compounds which effectively inhibit the wild-type enzyme, and had little effect on weak or moderate inhibitors. As a consequence, all compounds had similar inhibitory potencies to the substituted enzyme variants. An exception was VX-950, which inhibited the D168V enzyme more efficiently than the wild type. For this inhibitor, the present data correlated better with replicon data than data from assays with truncated enzyme.

    Conclusions: These results have provided a structural basis for designing inhibitors that may be less susceptible to resistance by three known mutations, and suggest that the present variants of full-length NS3 constitute effective models for resistance profiling of NS3 protease inhibitors.

  • 29.
    Danielson, Helena
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Lindgren, Maria T.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Markgren, Per-Olof
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Nillroth, Ulrika
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Investigation of an allosteric site of HIV-1 proteinase involved in inhibition by Cu2+1998In: Advances in Experimental Medicine and Biology, ISSN 0065-2598, E-ISSN 2214-8019, Vol. 436, 99-103 p.Article in journal (Refereed)
  • 30.
    Danielson, U Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Fragment library screening and lead characterization using SPR biosensors2009In: Current Topics in Medicinal Chemistry, ISSN 1568-0266, E-ISSN 1873-4294, Vol. 9, no 18, 1725-1735 p.Article in journal (Refereed)
    Abstract [en]

    The transition from high throughput screening of collections of drug-like compounds to screening of fragment libraries via lower throughput methods with high sensitivity has revolutionized early drug discovery. It is highlighting the need for sensitive biophysical techniques for interaction analysis rather than high throughput methods. Biosensors with SPR detection are well suited for this novel scenario. In less than 20 years the technique has been launched, established and become a highly informative method for a variety of applications in drug discovery. It is no longer limited to the detection of proteins or other high molecular weight analytes, but the detection of weakly interacting fragments is now feasible. This paper discusses the theoretical and experimental limitations for such applications and reviews a number of successful studies in the area of fragment-based lead discovery that have recently been published. It can be anticipated that the evolution of this young technique will be significantly influenced by the requirements for efficient fragment-based lead discovery.

  • 31.
    Danielson, U Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Integrating surface plasmon resonance biosensor-based interaction kinetic analyses into the lead discovery and optimization process2009In: Future Medicinal Chemistry, ISSN 1756-8919, E-ISSN 1756-8927, Vol. 1, 1399-1414 p.Article in journal (Refereed)
  • 32.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Molecular Interaction Analysis for Discovery of Drugs Targeting Enzymes and for Resolving Biological Function2015In: Multifaceted Roles Of Crystallography In Modern Drug Discovery / [ed] Scapin, G; Patel, D; Arnold, E, 2015, 223-240 p.Conference paper (Refereed)
    Abstract [en]

    Analysis of molecular interactions using surface plasmon resonance (SPR) biosensor technology has become a powerful tool for discovery of drugs targeting enzymes and resolving biological function. A major advantage of this technology over other methods for interaction analysis is that it can provide the kinetic details of interactions. This is a consequence of the time resolution of the analysis, which allows individual kinetic rate constants as well as affinities to be determined. A less commonly recognized feature of this technology is that it can reveal the characteristics of more complex mechanisms, e.g. involving multiple steps or conformations of the target or ligand, as well as the energetics, thermodynamics and forces involved.

  • 33.
    Danielson, U Helena
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Esterbauer, Hermann
    Mannervik, Bengt
    Structure-activity relationships of 4-hydroxyalkenals in the conjugation catalysed by mammalian glutathione transferases1987In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 247, no 3, 707-713 p.Article in journal (Refereed)
    Abstract [en]

    The substrate specificities of 15 cytosolic glutathione transferases from rat, mouse and man have been explored by use of a homologous series of 4-hydroxyalkenals, extending from 4-hydroxypentenal to 4-hydroxypentadecenal. Rat glutathione transferase 8-8 is exceptionally active with the whole range of 4-hydroxyalkenals, from C5 to C15. Rat transferase 1-1, although more than 10-fold less efficient than transferase 8-8, is the second most active transferase with the longest chain length substrates. Other enzyme forms showing high activities with these substrates are rat transferase 4-4 and human transferase mu. The specificity constants, kcat./Km, for the various enzymes have been determined with the 4-hydroxyalkenals. From these constants the incremental Gibbs free energy of binding to the enzyme has been calculated for the homologous substrates. The enzymes responded differently to changes in the length of the hydrocarbon side chain and could be divided into three groups. All glutathione transferases displayed increased binding energy in response to increased hydrophobicity of the substrate. For some of the enzymes, steric limitations of the active site appear to counteract the increase in binding strength afforded by increased chain length of the substrate. Comparison of the activities with 4-hydroxyalkenals and other activated alkenes provides information about the active-site properties of certain glutathione transferases. The results show that the ensemble of glutathione transferases in a given species may serve an important physiological role in the conjugation of the whole range of 4-hydroxyalkenals. In view of its high catalytic efficiency with all the homologues, rat glutathione transferase 8-8 appears to have evolved specifically to serve in the detoxication of these reactive compounds of oxidative metabolism.

  • 34.
    Danielson, U Helena
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Huang, H H
    Seeger, Christian
    Lindblad, Peter
    Analysis of the leakage of gene repression by an artificial TetR-regulated promoter in cyanobacteria2015In: BMC Research Notes, ISSN 1756-0500, E-ISSN 1756-0500, Vol. 8, 459- p.Article in journal (Refereed)
  • 35.
    Danielson, U. Helena
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Jiang, Fanyi Y
    Hansson, Lars O.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Probing the kinetic mechanism and coenzyme specificity of glutathione reductase from the cyanobacterium Anabaena PCC 7120 by redesign of the pyridine-nucleotide-binding site1999In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 38, no 29, 9254-9263 p.Article in journal (Refereed)
    Abstract [en]

    Glutathione reductase from the cyanobacterium Anabaena PCC 7120 contains a pyridine-nucleotide-binding motif differing from that of the enzyme from other sources and an insertion of 10 amino acid residues. Homology modeling was used to obtain a model of the enzyme structure. It revealed that in the Anabaena enzyme Lys(203) replaces Arg, found to interact with the 2'-phosphate of NADP(H) in the enzyme from other sources, and that it has an extra loop near the entrance of the pyridine-nucleotide-binding site. The steady-state and preequilibrium kinetic properties were characterized for the wild-type enzyme, a K203R, and a loop deletion mutant. All enzyme forms had higher catalytic efficiency with NADPH than with NADH, although the difference was less than for glutathione reductase from other sources. The specificity was most pronounced in the formation of the charge-transfer complex between the pyridine nucleotide and oxidized enzyme-bound FAD, as compared to later steps in the reaction. Unexpectedly, by replacing Lys(203) with Arg, the specificity for NADPH was diminished in the complete redox reaction. Ser(174) appears to interact with the 2'-phosphate of NADPH and introduction of arginine instead of lysine, therefore, has little effect on the interaction with this coenzyme. However, the efficiency in forming the charge-transfer complex between the pyridine nucleotide and oxidized enzyme-bound FAD was increased in the K203R mutant using NADPH but not with NADH. The lack of affinity toward 2',5'-ADP-Sepharose by the wild-type enzyme was not changed by replacing Lys(203) with Arg but deletion of the loop resulted in an enzyme that bound to the immobilized ligand. Removal of the loop increased the efficiency of the enzyme in the reductive half-reaction with both pyridine-nucleotides as well as in the overall catalytic mechanism.

  • 36.
    Danielson, U Helena
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mannervik, Bengt
    Kinetic independence of the subunits of cytosolic glutathione transferase from the rat1985In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 231, no 2, 263-267 p.Article in journal (Refereed)
    Abstract [en]

    The steady-state kinetics of the dimeric glutathione transferases deviate from Michaelis-Menten kinetics, but have hyperbolic binding isotherms for substrates and products of the enzymic reaction. The possibility of subunit interactions during catalysis as an explanation for the rate behaviour was investigated by use of rat isoenzymes composed of subunits 1, 2, 3 and 4, which have distinct substrate specificities. The kinetic parameter kcat./Km was determined with 1-chloro-2,4-dinitrobenzene, 4-hydroxyalk-2-enals, ethacrynic acid and trans-4-phenylbut-3-en-2-one as electrophilic substrates for six isoenzymes: rat glutathione transferases 1-1, 1-2, 2-2, 3-3, 3-4 and 4-4. It was found that the kcat./Km values for the heterodimeric transferases 1-2 and 3-4 could be predicted from the kcat./Km values of the corresponding homodimers. Likewise, the initial velocities determined with transferases 3-3, 3-4 and 4-4 at different degrees of saturation with glutathione and 1-chloro-2,4-dinitrobenzene demonstrated that the kinetic properties of the subunits are additive. These results show that the subunits of glutathione transferase are kinetically independent.

  • 37.
    Danielson, U Helena
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mannervik, Bengt
    Paradoxical inhibition of rat glutathione transferase 4-4 by indomethacin explained by substrate-inhibitor-enzyme complexes in a random-order sequential mechanism1988In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 250, no 3, 705-711 p.Article in journal (Refereed)
    Abstract [en]

    Under standard assay conditions, with 1-chloro-2,4-dinitrobenzene (CDNB) as electrophilic substrate, rat glutathione transferase 4-4 is strongly inhibited (I50 = 1 microM) by indomethacin. No other glutathione transferase investigated is significantly inhibited by micromolar concentrations of indomethacin. Paradoxically, the strong inhibition of glutathione transferase 4-4 was dependent on high (millimolar) concentrations of CDNB; at low concentrations of this substrate or with other substrates the effect of indomethacin on the enzyme was similar to the moderate inhibition noted for other glutathione transferases. In general, the inhibition of glutathione transferases can be explained by a random-order sequential mechanism, in which indomethacin acts as a competitive inhibitor with respect to the electrophilic substrate. In the specific case of glutathione transferase 4-4 with CDNB as substrate, indomethacin binds to enzyme-CDNB and enzyme-CDNB-GSH complexes with an even greater affinity than to the corresponding complexes lacking CDNB. Under presumed physiological conditions with low concentrations of electrophilic substrates, indomethacin is not specific for glutathione transferase 4-4 and may inhibit all forms of glutathione transferase.

  • 38. de Kloe, Gerdien E
    et al.
    Retra, Kim
    Geitmann, Matthis
    Källblad, Per
    Nahar, Tariq
    van Elk, René
    Smit, August B
    van Muijlwijk-Koezen, Jacqueline E
    Leurs, Rob
    Irth, Hubertus
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    de Esch, Iwan J P
    Surface Plasmon Resonance Biosensor Based Fragment Screening Using Acetylcholine Binding Protein Identifies Ligand Efficiency Hot Spots (LE Hot Spots) by Deconstruction of Nicotinic Acetylcholine Receptor α7 Ligands2010In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 53, no 19, 7192-7201 p.Article in journal (Refereed)
    Abstract [en]

    The soluble acetylcholine binding protein (AChBP) is a homologue of the ligand-binding domain of the nicotinic acetylcholine receptors (nAChR). To guide future fragment-screening using surface plasmon resonance (SPR) biosensor technology as a label-free, direct binding, biophysical screening assay, a focused fragment library was generated based on deconstruction of a set of α7 nAChR selective quinuclidine containing ligands with nanomolar affinities. The interaction characteristics of the fragments and the parent compounds with AChBP were evaluated using an SPR biosensor assay. The data obtained from this direct binding assay correlated well with data from the reference radioligand displacement assay. Ligand efficiencies for different (structural) groups of fragments in the library were correlated to binding with distinct regions of the binding pocket, thereby identifying ligand efficiency hot spots (LE hot spots). These hot spots can be used to identity the most promising hit fragments in a large scale fragment library screen.

  • 39. Domínguez, José L
    et al.
    Christopeit, Tony
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Villaverde, M Carmen
    Gossas, Thomas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Otero, José M
    Nyström, Susanne
    Baraznenok, Vera
    Lindström, Erik
    Danielson, U Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Sussman, Fredy
    Effect of the Protonation State of the Titratable Residues on the Inhibitor Affinity to BACE-12010In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 49, no 34, 7255-7263 p.Article in journal (Refereed)
    Abstract [en]

    BACE-1 is one of the aspartic proteases involved in the cleavage of beta amyloid peptide, an initial step in the formation of amyloid plaques whose toxicity induces neuron death in Alzheimer's disease patients. One of the central issues in the search of novel BACE-1 inhibitors is the optimum pH for the binding of inhibitors to the enzyme. It is known that the enzyme has optimal catalytic activity at acidic pH, while cell active inhibitors may bind optimally at higher pH. In this work we determine the effect of the pH on the affinities of a set of inhibitors, with a variety of chemical motifs, for the ectodomain region of BACE-1 by a surface plasmon resonance (SPR) biosensor based assay. In order to understand the molecular interactions that underlie the diverse optimum pH for the binding of the various inhibitors as observed experimentally, we have calculated the titration curves for a set of BACE-1 ligand complexes. The results indicate that the pK(a) values of the titratable residues of the protein depend on the nature of the ligand involved, in disagreement with previous work. The enzyme-inhibitor structures with the resulting protonation states at pH values 4.5 and 7.4 served as the starting point for the prediction of the pH-dependent binding ranking. Our calculations reproduced the entire affinity ranking observed upon pH increase and most of the binding trends among inhibitors, especially at low pH. Finally, our cell-based assays indicate a possible correlation between high inhibitor affinity at both acidic and neutral pH values, with optimal cell response, a result that may open new venues for the search of potent BACE-1 inhibitors that are active at the cellular level.

  • 40. Dominguez, Jose L.
    et al.
    Gossas, Thomas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Carmen Villaverde, M.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Sussman, Fredy
    Experimental and 'in silico' analysis of the effect of pH on HIV-1 protease inhibitor affinity: Implications for the charge state of the protein ionogenic groups2012In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 20, no 15, 4838-4847 p.Article in journal (Refereed)
    Abstract [en]

    The pH dependence of the HIV-1 protease inhibitor affinity was studied by determining the interaction kinetics of a series of inhibitors at three pH values by surface plasmon resonance (SPR) biosensor analysis. The results were rationalized by molecular mechanics based protocols that have as a starting point the structures of the HIV-1 protease inhibitor complexes differing in the protonation states as predicted by our calculations. The SPR experiments indicate a variety of binding affinity pH dependencies which are rather well reproduced by our simulations. Moreover, our calculations are able to pinpoint the possible changes in the charged state of the protein binding site and of the inhibitor that underlie the observed effects of the pH on binding affinity. The combination of SPR and molecular mechanics calculations has afforded novel insights into the pH dependence of inhibitor interactions with their target. This work raises the possibility of designing inhibitors with different pH binding affinity profiles to the ones described here.

  • 41.
    Ehrenberg, Angelica
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Schmuck, Benjamin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Anwar, Muhammad Ikram
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Svahn Gustafsson, Sofia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Stenberg, Gun
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Danielson, Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Accounting for strain variations and resistance mutations in the characterization of hepatitis C NS3 protease inhibitors2014In: Journal of enzyme inhibition and medicinal chemistry (Print), ISSN 1475-6366, E-ISSN 1475-6374, Vol. 29, no 6, 868-876 p.Article in journal (Refereed)
    Abstract [en]

    Context: Natural strain variation and rapid resistance development makes development of broad spectrum hepatitis C virus (HCV) drugs very challenging and evaluation of inhibitor selectivity and resistance must account for differences in the catalytic properties of enzyme variants.

    Objective: To understand how to study selectivity and relationships between efficacy and genotype or resistant mutants for NS3 protease inhibitors.

    Materials and methods: The catalytic properties of NS3 protease from genotypes 1a, 1b and 3a, and their sensitivities to four structurally and mechanistically different NS3 protease inhibitors have been analysed under different experimental conditions.

    Results: The optimisation of buffer conditions for each protease variant enabled the comparison of their catalytic properties and sensitivities to the inhibitors. All inhibitors were most effective against genotype 1a protease, with VX-950 having the broadest selectivity.

    Discussion and conclusion: A new strategy for evaluation of inhibitors relevant for the discovery of broad spectrum HCV drugs was established.

  • 42.
    Elinder, Malin
    et al.
    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.
    Unge, Torsten
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    VanHam, Guido
    Öberg, Bo
    MIV-170: A novel NNRTI exhibiting tight binding to HIV-1 reverse transcriptase (RT)2008Conference paper (Refereed)
    Abstract [en]

    The NNRTI MIV-170 has been found to be a very efficient inhibitor of wtHIV and HIV mutant strains resistant to the NNRTIs used in the clinic. To better understand the interaction between MIV-170 and HIV-1 the details of this have been studied by different methods. The kinetics of the interaction between MIV-170 and HIV-1 RT was analysed using a biosensor assay. The association and dissociation rates were determined using immobilized wtRT or RT mutants and MIV-170 as analyte. The results demonstrated that MIV-170 had both a faster association and a slower dissociation rate than efavirenz, nevirapine and delavirdine, thus exhibiting a higher affinity than these compounds. The strength of the interaction between the NNRTIs and RT and RT mutants in the biosensor assay was compared to the reversibility of inhibition in cell culture experiments. In these experiments virus and infected cells were incubated with MIV-170 and other NNRTIs for various times and after removal of the compounds the remaining infectivity was assayed. X-ray analysis of the binding of MIV-170 to HIV-1 RT displayed extensive interactions, not only between the compound and the lining amino acids but also between these residues, turning the binding cavity into a rigid entity and explaining the tight binding in the biosensor assay and the inactivation of HIV.

  • 43. Elinder, Malin
    et al.
    Danielson, U Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Unge, Torsten
    van Ham, Guido
    Öberg, Bo
    MIV-170, a novel NNRTI exhibiting tight binding to HIV-1 reverse transcriptase (RT)2008In: Antiviral Research, ISSN 0166-3542, E-ISSN 1872-9096, Vol. 78, A37-A37 p.Article in journal (Refereed)
  • 44.
    Elinder, Malin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Geitmann, Matthis
    Gossas, Thomas
    Källblad, Per
    Winquist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Nordström, Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Hämäläinen, Markkuu D.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Experimental Validation of a Fragment Library for Lead Discovery Using SPR Biosensor Technology2011In: Journal of Biomolecular Screening, ISSN 1087-0571, E-ISSN 1552-454X, Vol. 16, no 1, 15-25 p.Article in journal (Refereed)
    Abstract [en]

    A new fragment library for lead discovery has been designed and experimentally validated for use in surface plasmon resonance (SPR) biosensor-based screening. The 930 compounds in the library were selected from 4.6 million commercially available compounds using a series of physicochemical and medicinal chemistry filters. They were screened against 3 prototypical drug targets: HIV-1 protease, thrombin and carbonic anhydrase, and a nontarget: human serum albumin. compound solubility was not a problem under the conditions used for screening. The high sensitivity of the sensor surfaces allowed the detection of interactions for 35% to 97% of the fragments, depending on the target protein. None of the fragments was promiscuous (i.e., interacted with a stoichiometry ≥5:1 with all 4 proteins), and only 2 compounds dissociated slowly from all 4 proteins. The use of several targets proved valuable since several compounds would have been disqualified from the library on the grounds of promiscuity if fewer target proteins had been used. The experimental procedure allowed an efficient evaluation and exploration of the new fragment library and confirmed that the new library is suitable for SPR biosensor-based screening.

  • 45.
    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, 395-403 p.Article 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.

  • 46.
    Elinder, Malin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Selhorst, Philippe
    Vanham, Guido
    Öberg, Bo
    Vrang, Lotta
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Inhibition of HIV-1 by non-nucleoside reverse transcriptase inhibitors via an induced fit mechanism: Importance of slow dissociation and relaxation rates for antiviral efficacy2010In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 80, no 8, 1133-1140 p.Article in journal (Refereed)
    Abstract [en]

    The importance of slow dissociation of non-nucleoside reverse transcriptase inhibitors (NNRTIs) for antiviral effect has been investigated. The kinetic characteristics of a series of NNRTIs interacting with wild type and drug resistant variants of HIV-1 RT (EC 2.7.7.49) were analyzed by SPR biosensor technology. The antiviral effect was determined in MT-4 and peripheral blood mononuclear cells. Due to extremely slow dissociation rates and a complex interaction mechanism, rate constants could not be quantified. Instead, interaction characteristics were qualitatively analyzed using simulated sensorgrams. The simplest model describing these interactions adequately was an induced fit mechanism, i.e. a mechanism involving the formation of an initial enzyme-inhibitor complex subsequently transformed into a more stable complex. Differences in rates of dissociation from the initial complex and rates of relaxation from the induced complex explained (1) the differences in the amounts of formed complex, (2) the stability of the complex and (3) the antiviral efficacies of the compounds. The effect of NNRTI binding site mutations also correlated with these kinetic characteristics. MIV-170 was the most effective inhibitor of wild type and mutant HIV-1 in cell culture, a property that was associated with the formation of the largest amount of complex and the slowest relaxation and dissociation rates. This study supports the hypothesis that the efficacy of anti-HIV drugs is dependent on slow dissociation from the target, thereby maximizing the duration of the inhibitory effect. It also illustrates the strength of simulating interaction data for qualitative analysis of tight-binding drugs and the importance of resolving the kinetic mechanism of drug-target interactions.

  • 47.
    Fabini, Edoardo
    et al.
    Univ Bologna, Dept Pharm & Biotechnol, Via Belmeloro 6, I-40126 Bologna, Italy..
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Monitoring drug-serum protein interactions for early ADME prediction through Surface Plasmon Resonance technology2017In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 144, 188-194 p.Article in journal (Refereed)
    Abstract [en]

    Many molecules fail to reach the market due to poor pharmacokinetic (PK) properties, rendering the potential drug virtually unavailable for the primary target despite efficient administration to the body. PK properties of endogenous and exogenous compounds in mammals are dependent, among other factors, on their ability to interact with serum proteins. The extent of binding can greatly influence their ADME (adsorption, distribution, metabolism and execration) profile. Reliable and cost-effective bioavailability studies, early in the drug discovery process, can lead to an improvement of the success rate for compounds entering clinical trials. Optical biosensors based on surface plasmon resonance (SPR) detection emerged as an efficient approach to obtain large amounts of information about the binding of small molecules to serum proteins. Simple, automated and fast assays provide a good throughput, versatility and highly informative data output, rendering the methodology particularly suited for early screening. The ability to provide basic information on PK can be easily coupled to structure-activity relationship analysis. In this review, features of the technology and its employment for the study of serum protein-small molecule interactions are presented and discussed. 

  • 48.
    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, Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Kinetic characterization of HCV NS3 inhibitors using an SPR biosensorManuscript (Other academic)
    Abstract [en]

    An SPR biosensor-based assay for studies of the interactions with full-length NS3 protease from hepatitis C virus (HCV) has been developed. It was used to characterize the interaction kinetics for a series of NS3 protease inhibitors. Moreover, the interaction between NS3 and the NS4A cofactor could be studied.

    The KD of the NS3-NS4A interaction was 30 nM in the standard buffer. It was reduced 600-fold by increasing the ionic strength to 300 mM NaCl. By using surfaces with only NS3 or with NS3 and co-immobilised NS4A, the effect of this protein cofactor on the interaction with several protease inhibitors was investigated. NS4A increased the affinity for all compounds, between 2 to 40 times, indicating that the NS3-NS4A complex binds inhibitors better than only NS3. The obtained interaction data was also compared with inhibition data, revealing a very good correlation between koff or KD with Ki (r = 0.92 and r = 0.90 respectively) over a broad range of affinities and potencies, showing that this biosensor based assay is a good and powerful tool for detailed studies of NS3 protease inhibitors which can serve as a future cure for HCV infection.

  • 49.
    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, 60-70 p.Article 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.

  • 50.
    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.
    Lohmann, Volker
    Department of Molecular Virology, University of Heidelberg, Germany.
    Paeshuyse, Jan
    Rega Institute, Leuven, Belgium.
    Leyssen, Pieter
    Rega Institute, Leuven, Belgium.
    Herdewijn, Piet
    Rega Institute, Leuven, Belgium.
    Puerstinger, Gerhard
    Institute of Pharmacy, University of Innsbruck, Innsbruck, Austria.
    Bartenschlager, Ralf
    Department of Molecular Virology, University of Heidelberg, Germany.
    Neyts, Johan
    Rega Institute, Leuven, Belgium.
    Danielson, Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Kinetic, mechanistic and chemodynamic characterisation of non-nucleoside hepatitis C virus NS5B polymerase inhibitors using SPR biosensor technologyManuscript (Other academic)
    Abstract [en]

    Kinetic, mechanistic and chemodynamic aspects of the interaction between five non-nucleoside inhibitors and the HCV NS5B polymerase (genotype 2a) were assessed using SPR biosensor technology. The compounds were selected to represent different structural classes (benzothiadiazine, , α,γ-diketo acid, benzimidazole, thiophene carbocyclic acid and benzofuran), each known to interact with different binding sites. The viral polymerase interacted with the compounds with different kinetics and surprisingly also with different capacities. Cooperativity between the different allosteric inhibitor binding sites and the active site binding diketoacid was observed, but no cooperativity was seen between the allosteric sites. The interaction with diketoacid was stronger in phosphate buffer as compared to Tris buffer, indicating a phosphate ion-mediated interaction mechanism. The enzyme generally had reduced affinity for the inhibitors in the presence of RNA. Interaction parameters determined for human serum albumin revealed the propensity of the compounds to be distributed by HSA. This study provides important information for the design of optimized NS5B inhibitors and illustrates the complementarity of a biosensor-based analysis with inhibition studies, in particular for allosteric compounds with complex interaction mechanisms or when the target contains multiple ligand binding sites.

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