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  • 1.
    Abdurakhmanov, Eldar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Discovery and evaluation of direct acting antivirals against hepatitis C virus2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Until recently, the standard therapy for hepatitis C treatment has been interferon and ribavirin. Such treatment has only 50% efficacy and is not well tolerated. The emergence of new drugs has increased the treatment efficacy to 90%. Despite such an achievement, the success is limited since the virus mutates rapidly, causing the emergence of drug resistant forms. In addition, most new drugs were developed to treat genotype 1 infections. Thus, development of new potent antivirals is needed and drug discovery against hepatitis C is continued.

    In this thesis, a FRET-based protease assay was used to evaluate new pyrazinone based NS3 protease inhibitors that are structurally different to the newly approved and currently developing drugs. Several compounds in this series showed good potencies in the nanomolar range against NS3 proteases from genotype 1, 3, and the drug resistance variant R155K. We assume that these compounds can be further developed into drug candidates that possess activity against above mentioned enzyme variants.

    By using SPR technology, we analyzed interaction mechanisms and characteristics of allosteric inhibitors targeting NS5B polymerases from genotypes 1 and 3. The compounds exhibited different binding mechanisms and displayed a low affinity against NS5B from genotype 3.

    In order to evaluate the activity and inhibitors of the NS5B polymerase, we established an SPR based assay, which enables the monitoring of polymerization and its inhibition in real time. This assay can readily be implemented for the discovery of inhibitors targeting HCV.

    An SPR based fragment screening approach has also been established. A screen of a fragment library has been performed in order to identify novel scaffolds that can be used as a starting point for development of new allosteric inhibitors against NS5B polymerase. Selected fragments will be further elaborated to generate a new potent allosteric drug candidate.

    Alternative approaches have successfully been developed and implemented to the discovery of potential lead compounds targeting two important HCV drug targets.

    List of papers
    1. Discovery of pyrazinone based compounds that potently inhibit the drug resistant enzyme variant R155K of the hepatitis C virus NS3 protease
    Open this publication in new window or tab >>Discovery of pyrazinone based compounds that potently inhibit the drug resistant enzyme variant R155K of the hepatitis C virus NS3 protease
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    2016 (English)In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 24, no 12, p. 2603-2620Article in journal (Refereed) Published
    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.

    Keywords
    Hepatitis C virus; Drug resistance; Pyrazinone; NS3 protease inhibitors; R155K
    National Category
    Organic Chemistry
    Research subject
    Medicinal Chemistry
    Identifiers
    urn:nbn:se:uu:diva-243315 (URN)10.1016/j.bmc.2016.03.066 (DOI)000376727800002 ()27160057 (PubMedID)
    Funder
    Swedish Research Council, D0571301
    Available from: 2015-02-08 Created: 2015-02-08 Last updated: 2017-12-04Bibliographically approved
    2. Pyrazinone based hepatitis C virus NS3 protease inhibitors targeting genotype 1a, 3a and the drug-resistant enzyme variant R155K
    Open this publication in new window or tab >>Pyrazinone based hepatitis C virus NS3 protease inhibitors targeting genotype 1a, 3a and the drug-resistant enzyme variant R155K
    Show others...
    (English)Manuscript (preprint) (Other academic)
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-265295 (URN)
    Available from: 2015-10-26 Created: 2015-10-26 Last updated: 2016-01-13
    3. Resolution of the Interaction Mechanisms and Characteristics of Non-nucleoside Inhibitors of Hepatitis C Virus Polymerase - Laying the Foundation for Discovery of Allosteric HCV Drugs
    Open this publication in new window or tab >>Resolution of the Interaction Mechanisms and Characteristics of Non-nucleoside Inhibitors of Hepatitis C Virus Polymerase - Laying the Foundation for Discovery of Allosteric HCV Drugs
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    2013 (English)In: Antiviral Research, ISSN 0166-3542, E-ISSN 1872-9096, Vol. 97, no 3, p. 356-368Article in journal (Other academic) Published
    Abstract [en]

    Development of allosteric inhibitors into efficient drugs is hampered by their indirect mode-of-action and complex structure-kinetic relationships. To enablethe design of efficient allosteric drugs targeting the polymerase of hepatitis C virus(NS5B), the interaction characteristics of three non-nucleoside compounds (filibuvir, VX-222, and tegobuvir) inhibiting HCV replication via NS5B have been analyzed. Since there was no logical correlation between the anti-HCV replicative and enzyme inhibitory effects of the compounds, surface plasmon resonance biosensor technology was used to resolve the mechanistic, kinetic, thermodynamic and chemodynamic features of their interactions with their target and their effect on itsinteraction with RNA. Tegobuvir could not be seen to interact with NS5B at all while filibuvir interacted in a single reversible step (except at low temperatures) and VX-222 in two serial steps, interpreted as an induced fit mechanism. Both filibuvir and VX-222 interfered with the interaction between NS5B and RNA. They competed for binding to the enzyme, suggesting that they had a common inhibition mechanism and identical or overlapping binding sites. The greater anti-HCV replicative activityof VX-222 over filibuvir is hypothesized to be due to a greater allosteric conformational effect, resulting in the formation of a less catalytically competent complex. In addition, the induced fit mechanism of VX-222 gives it a kinetic advantage over filibuvir, exhibited as a longer residence time. These insights have important consequences for the selection and optimization of new allosteric NS5Binhibitors.

    Keywords
    HCV, NS5B, filibuvir, VX-222, tegobuvir, allosteric inhibitor, induced fit, kinetics, chemodynamics, thermodynamics
    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biochemistry; Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-171996 (URN)10.1016/j.antiviral.2012.12.027 (DOI)000317709400018 ()
    Available from: 2012-04-03 Created: 2012-03-31 Last updated: 2017-12-07Bibliographically approved
    4. Characterization of allosteric inhibitors of hepatitis C virus polymerase – a genotype comparative study
    Open this publication in new window or tab >>Characterization of allosteric inhibitors of hepatitis C virus polymerase – a genotype comparative study
    (English)Manuscript (preprint) (Other academic)
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-265287 (URN)
    Available from: 2015-10-26 Created: 2015-10-26 Last updated: 2016-01-13
    5. A time-resolved surface plasmon resonance based hepatitis C virus NS5B polymerase assay and its application for drug discovery
    Open this publication in new window or tab >>A time-resolved surface plasmon resonance based hepatitis C virus NS5B polymerase assay and its application for drug discovery
    (English)Manuscript (preprint) (Other academic)
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-265290 (URN)
    Available from: 2015-10-26 Created: 2015-10-26 Last updated: 2016-01-13
    6. Fragment library screening addressing Hepatitis C protein NS5B from genotypes 1 and 3 using an SPR-based approach
    Open this publication in new window or tab >>Fragment library screening addressing Hepatitis C protein NS5B from genotypes 1 and 3 using an SPR-based approach
    Show others...
    (English)Manuscript (preprint) (Other academic)
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-265292 (URN)
    Available from: 2015-10-26 Created: 2015-10-26 Last updated: 2016-01-13
  • 2.
    Abdurakhmanov, Eldar
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Solbak, Sara
    Danielson, Helena
    Characterization of allosteric inhibitors of hepatitis C virus polymerase – a genotype comparative studyManuscript (preprint) (Other academic)
  • 3.
    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, article id 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.

  • 4.
    Aksoy, N. H.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Aksaray Univ, Dept Biochem, Aksaray, Turkey..
    Mannervik, B.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Inhibitory effects of ethacrynic acid on glutathione S-transferase A1-1 from Callithrix jacchus2015In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 282, p. 348-348Article in journal (Other academic)
  • 5.
    Al-Amin, Rasel A.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Science for Life Laboratory, SciLifeLab, Science for Life Laboratory, SciLifeLab.
    Johansson, Lars
    Division of Translational Medicine & Chemical Biology, Department of Medical Biochemistry & Biophysics, Karolinska Institutet.
    Landegren, Nils
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Autoimmunity. Uppsala University, Science for Life Laboratory, SciLifeLab. Department of Medicine (Solna), Karolinska University Hospital, Karolinska Institutet.
    Löf, Liza
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Abdurakhmanov, Eldar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Blokzijl, Andries
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Svensson, Richard
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lönn, Peter
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Dept. Of Immunology, Genetics and Pathology,.
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Artursson, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lundbäck, Thomas
    Division of Translational Medicine & Chemical Biology, Department of Medical Biochemistry & Biophysics, Karolinska Institutet.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Target Engagement-Mediated Amplification for Monitoring Drug-Target Interactions in SituManuscript (preprint) (Other academic)
    Abstract [en]

    It is important to determine the localization of drugs or drug candidates at cellular and subcellular resolution in relevant clinical specimens. This is necessary to evaluate drug candidates from early stages of drug development to clinical evaluation of mutations potentially causing resistance to targeted therapy. We describe a technology where oligonucleotide-conjugated drug molecules are used to visualize and measure target engagement in situ via rolling-circle amplification (RCA) of circularized oligonucleotide probes (padlock probes). We established this target engagement-mediated amplification (TEMA) technique using kinase inhibitor precursor compounds, and we applied the assay to investigate target interactions by microscopy in pathology tissue sections and using flow cytometry for blood samples from patients, as well as in commercial arrays including almost half of all human proteins.  In the variant proxTEMAtechnique, in situ proximity ligation assays were performed by combining drug-DNA conjugates with antibody-DNA conjugates to specifically reveal drug binding to particular on- or off-targets in pathological tissues sections. In conclusion, the TEMA methods successfully visualize drug-target interaction by experimental and clinically approved kinase inhibitors in situ and with kinases among a large collection of arrayed proteins. 

  • 6.
    Ali, Muhammad
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    High-throughput discovery of functional disordered regions2018In: Molecular Systems Biology, ISSN 1744-4292, E-ISSN 1744-4292, Vol. 14, no 5, article id e8377Article in journal (Other academic)
    Abstract [en]

    Partially or fully intrinsically disordered proteins are widespread in eukaryotic proteomes and play important biological functions. With the recognition that well defined protein structure is not a fundamental requirement for function come novel challenges, such as assigning function to disordered regions. In their recent work, Babu and colleagues (Ravarani etal,) took on this challenge by developing IDR-Screen, a robust high-throughput approach for identifying functions of disordered regions.

  • 7.
    Al-Smadi, Derar
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry. An Najah Natl Univ, Dept Chem, Fac Sci, Nablus, Palestine.
    Enugala, Thilak Reddy
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Kessler, Vadim
    Swedish Univ Agr Sci, Dept Mol Sci, Box 7015, SE-75007 Uppsala, Sweden.
    Mhasal, Anil Rhanu
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Kamerlin, Shina Caroline Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Kihlberg, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Norberg, Thomas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Chemical and Biochemical Approaches for the Synthesis of Substituted Dihydroxybutanones and Di-, and Tri-Hydroxypentanones2019In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 84, no 11, p. 6982-6991Article in journal (Refereed)
    Abstract [en]

    Polyhydroxylated compounds are building blocks for the synthesis of carbohydrates and other natural products. Their synthesis is mainly achieved by different synthetic versions of aldol-coupling reactions, catalyzed either by organocatalysts, enzymes or metal-organic catalysts. We have investigated the formation of 1,4-substituted 2,3-dihydroxybutan-1-one derivatives from para- and meta-substituted phenylacetaldehydes by three distinctly different strategies. The first involved a direct aldol reaction with hydroxyacetone, dihydroxyacetone or 2-hydroxyacetophenone, catalyzed by the cinchona derivative cinchonine. The second was reductive cross-coupling with methyl or phenyl glyoxal promoted by SmI2 resulting in either 5-substituted 3,4-dihydroxypentan-2-ones or 1,4 bis-phenyl substituted butanones, respectively. Finally, in the third case, aldolase catalysis was employed for synthesis of the corresponding 1,3,4-trihydroxylated pentan-2-one derivatives. The organocatalytic route with cinchonine generated distereomerically enriched syn products (de = 60−99 %), with moderate enantiomeric excesses (ee = 43−56%), but did not produce aldols with either hydroxyacetone or dihydroxyacetone as donor ketones. The SmI2-promoted reductive cross-coupling generated product mixtures with diastereomeric and enantiomeric ratios close to unity. This route allowed for the production of both 1-methyl- and 1-phenylsubstituted 2,3-dihydroxybutanones, at yields between 40−60%. Finally, the biocatalytic approach resulted in enantiopure syn (3R,4S) 1,3,4-trihydroxypentan-2-ones.

  • 8.
    al-smadi, Derar
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Enugala, Thilak Reddy
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Norberg, Thomas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Kessler, Vadim
    Kihlberg, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    A Comparison of Synthetic Approaches to Derivatives of 1,4-Substituted 2,3 DihydroxybutanonesManuscript (preprint) (Other academic)
  • 9.
    Al-Smadi, Derar
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Enugala, Thilak Reddy
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Norberg, Thomas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Kihlberg, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Synthesis of substrates for aldolase-catalyzed reactions: A comparison of methods for the synthesis of substituted phenylacetaldehydes2018In: Synlett: Accounts and Rapid Communications in Synthetic Organic Chemistry, ISSN 0936-5214, E-ISSN 1437-2096, Vol. 29, no 9, p. 1187-1190Article in journal (Refereed)
    Abstract [en]

    Methods for the synthesis of phenylacetaldehydes (oxidation, one-carbon chain extension) were compared by using the synthesis of 4-methoxyphenylacetaldehyde as a model example. Oxidations of 4-methoxyphenylethanol with activated DMSO (Swern oxidation) or manganese dioxide gave unsatisfactory results; whereas oxidation with 2-iodoxybenzoic add (IBX) produced 4-methoxyphenylacetaldehyde in reasonable (75%) yield. However, Wittig-type one-carbon chain extension with methoxymethylene-triphenylphosphine followed by hydrolysis gave an excellent (81% overall) yield of 4-methoxyphenylacetaldehyde from 4-methoxybenzaldehyde (a cheap starting material). This approach was subsequently used to synthesise a set of 10 substituted phenylacetaldehydes in good to excellent yields.

  • 10.
    Amrein, Beat A.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Bauer, Paul
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Duarte, Fernanda
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Janfalk Carlsson, Åsa
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Naworyta, Agata
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Mowbray, Sherry L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Kamerlin, Shina C. L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Expanding the catalytic triad in epoxide hydrolases and related enzymes2015In: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 5, no 10, p. 5702-5713Article in journal (Refereed)
    Abstract [en]

    Potato epoxide hydrolase 1 exhibits rich enantio- and regioselectivity in the hydrolysis of a broadrange of substrates. The enzyme can be engineered to increase the yield of optically pureproducts, as a result of changes in both enantio- and regioselectivity. It is thus highly attractive inbiocatalysis, particularly for the generation of enantiopure fine chemicals and pharmaceuticals.The present work aims to establish the principles underlying the activity and selectivity of theenzyme through a combined computational, structural, and kinetic study, using the substratetrans-stilbene oxide as a model system. Extensive empirical valence bond simulations have beenperformed on the wild-type enzyme together with several experimentally characterized mutants.We are able to computationally reproduce the differences in activities between differentstereoisomers of the substrate, and the effects of mutations in several active-site residues. Inaddition, our results indicate the involvement of a previously neglected residue, H104, which iselectrostatically linked to the general base, H300. We find that this residue, which is highlyconserved in epoxide hydrolases and related hydrolytic enzymes, needs to be in its protonatedform in order to provide charge balance in an otherwise negatively-charged active site. Our datashow that unless the active-site charge balance is correctly treated in simulations, it is notpossible to generate a physically meaningful model for the enzyme that can accurately reproduceactivity and selectivity trends. We also expand our understanding of other catalytic residues,demonstrating in particular the role of a non-canonical residue, E35, as a “backup-base” in theabsence of H300. Our results provide a detailed view of the main factors driving catalysis andregioselectivity in this enzyme, and identify targets for subsequent enzyme design efforts.

  • 11.
    Amrein, Beat Anton
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab.
    Runthala, Ashish
    Kamerlin, Shina C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    In Silico-Directed Evolution Using CADEE2018In: Computational Methods in Protein Evolution / [ed] T. Tobias Sikosek, Springer Science+Business Media, LLC, part of Springer Nature , 2018, p. 381-415Chapter in book (Other academic)
  • 12. 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, p. 2270-2282Article 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.

  • 13.
    Bauer, Paul
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Computational modelling of enzyme selectivity2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Enantioselective reactions are one of the ways to produce pure chiral compounds. Understanding the basis of this selectivity makes it possible to guide enzyme design towards more efficient catalysts. One approach to study enzymes involved in chiral chemistry is through the use of computational models that are able to simulate the chemical reaction taking place. The potato epoxide hydrolase is one enzyme that is known to be both highly enantioselective, while still being robust upon mutation of residues to change substrate scope. The enzyme was used to investigate the epoxide hydrolysis mechanism for a number of different substrates, using the EVB approach to the reaction both in solution and in several enzyme variants. In addition to this, work has been performed on new ways of performing simulations of divalent transition metals, as well as development of new simulation software.

    List of papers
    1. Force Field Independent Metal Parameters Using a Nonbonded Dummy Model
    Open this publication in new window or tab >>Force Field Independent Metal Parameters Using a Nonbonded Dummy Model
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    2014 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 118, no 16, p. 4351-4362Article in journal (Refereed) Published
    Abstract [en]

    The cationic dummy atom approach provides a powerful nonbonded description for a range of alkaline-earth and transition-metal centers, capturing both structural and electrostatic effects. In this work we refine existing literature parameters for octahedrally coordinated Mn2+, Zn2+, Mg2+, and Ca2+, as well as providing new parameters for Ni2+, Co2+, and Fe2+. In all the cases, we are able to reproduce both M2+-O distances and experimental solvation free energies, which has not been achieved to date for transition metals using any other model. The parameters have also been tested using two different water models and show consistent performance. Therefore, our parameters are easily transferable to any force field that describes nonbonded interactions using Coulomb and Lennard-Jones potentials. Finally, we demonstrate the stability of our parameters in both the human and Escherichia coli variants of the enzyme glyoxalase 1 as showcase systems, as both enzymes are active with a range of transition metals. The parameters presented in this work provide a valuable resource for the molecular simulation community, as they extend the range of metal ions that can be studied using classical approaches, while also providing a starting point for subsequent parametrization of new metal centers.

    National Category
    Physical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-225523 (URN)10.1021/jp501737x (DOI)000335113600010 ()
    Funder
    Swedish National Infrastructure for Computing (SNIC), 2013/26-1
    Available from: 2014-06-23 Created: 2014-06-04 Last updated: 2018-12-03Bibliographically approved
    2. Expanding the catalytic triad in epoxide hydrolases and related enzymes
    Open this publication in new window or tab >>Expanding the catalytic triad in epoxide hydrolases and related enzymes
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    2015 (English)In: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 5, no 10, p. 5702-5713Article in journal (Refereed) Published
    Abstract [en]

    Potato epoxide hydrolase 1 exhibits rich enantio- and regioselectivity in the hydrolysis of a broadrange of substrates. The enzyme can be engineered to increase the yield of optically pureproducts, as a result of changes in both enantio- and regioselectivity. It is thus highly attractive inbiocatalysis, particularly for the generation of enantiopure fine chemicals and pharmaceuticals.The present work aims to establish the principles underlying the activity and selectivity of theenzyme through a combined computational, structural, and kinetic study, using the substratetrans-stilbene oxide as a model system. Extensive empirical valence bond simulations have beenperformed on the wild-type enzyme together with several experimentally characterized mutants.We are able to computationally reproduce the differences in activities between differentstereoisomers of the substrate, and the effects of mutations in several active-site residues. Inaddition, our results indicate the involvement of a previously neglected residue, H104, which iselectrostatically linked to the general base, H300. We find that this residue, which is highlyconserved in epoxide hydrolases and related hydrolytic enzymes, needs to be in its protonatedform in order to provide charge balance in an otherwise negatively-charged active site. Our datashow that unless the active-site charge balance is correctly treated in simulations, it is notpossible to generate a physically meaningful model for the enzyme that can accurately reproduceactivity and selectivity trends. We also expand our understanding of other catalytic residues,demonstrating in particular the role of a non-canonical residue, E35, as a “backup-base” in theabsence of H300. Our results provide a detailed view of the main factors driving catalysis andregioselectivity in this enzyme, and identify targets for subsequent enzyme design efforts.

    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-260232 (URN)10.1021/acscatal.5b01639 (DOI)000362391500006 ()
    Funder
    EU, FP7, Seventh Framework Programme, 306474Swedish Research Council, 621-2011-6055, 621-2010-5145Swedish National Infrastructure for Computing (SNIC), 2015/16-12
    Available from: 2015-08-18 Created: 2015-08-18 Last updated: 2017-12-04Bibliographically approved
    3. Conformational Diversity and Enantioconvergence in Potato Epoxide Hydrolase 1
    Open this publication in new window or tab >>Conformational Diversity and Enantioconvergence in Potato Epoxide Hydrolase 1
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    2016 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 14, no 24, p. 5639-5651Article in journal (Refereed) Published
    Abstract [en]

    Potato epoxide hydrolase 1 (StEH1) is a biocatalytically important enzyme that exhibits rich enantio-and regioselectivity in the hydrolysis of chiral epoxide substrates. In particular, StEH1 has been demonstrated to enantioconvergently hydrolyze racemic mixes of styrene oxide (SO) to yield (R)-1-phenylethanediol. This work combines computational, crystallographic and biochemical analyses to understand both the origins of the enantioconvergent behavior of the wild-type enzyme, as well as shifts in activities and substrate binding preferences in an engineered StEH1 variant, R-C1B1, which contains four active site substitutions (W106L, L109Y, V141K and I155V). Our calculations are able to reproduce both the enantio-and regioselectivities of StEH1, and demonstrate a clear link between different substrate binding modes and the corresponding selectivity, with the preferred binding modes being shifted between the wild-type enzyme and the R-C1B1 variant. Additionally, we demonstrate that the observed changes in selectivity and the corresponding enantioconvergent behavior are due to a combination of steric and electrostatic effects that modulate both the accessibility of the different carbon atoms to the nucleophilic side chain of D105, as well as the interactions between the substrate and protein amino acid side chains and active site water molecules. Being able to computationally predict such subtle effects for different substrate enantiomers, as well as to understand their origin and how they are affected by mutations, is an important advance towards the computational design of improved biocatalysts for enantioselective synthesis.

    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-282015 (URN)10.1039/C6OB00060F (DOI)000378933400042 ()27049844 (PubMedID)
    Funder
    Swedish National Infrastructure for Computing (SNIC), 25/2-10EU, European Research Council, 306474;283570Swedish Research Council, 621-2011-6055Carl Tryggers foundation , CTS13:104
    Available from: 2016-04-01 Created: 2016-04-01 Last updated: 2017-11-30Bibliographically approved
    4. Laboratory evolved enzymes provide snapshots of the development of enantioconvergence in enzyme-catalyzed epoxide hydrolysis
    Open this publication in new window or tab >>Laboratory evolved enzymes provide snapshots of the development of enantioconvergence in enzyme-catalyzed epoxide hydrolysis
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    2016 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 17, no 18, p. 1693-1697Article in journal (Refereed) Published
    Abstract [en]

    Engineered enzyme variants of potato epoxide hydrolase (StEH1) display varying degrees of enrichment of (2R)-3-phenylpropane-1,2-diol from racemic benzyloxirane. Curiously, the observed increase in the enantiomeric excess of the (R)-diol is not only due to changes in enantioselectivity for the preferred epoxide enantiomer, but also to changes in the regioselectivity of the epoxide ring opening of (S)-benzyloxirane. To probe the structural origin of these differences in substrate selectivities and catalytic regiopreferences, we have solved the crystal structures for the in-vitro evolved StEH1 variants. We have additionally used these structures as a starting point for docking the epoxide enantiomers into the respective active sites. Interestingly, despite the simplicity of our docking calculations, the apparent preferred binding modes obtained from the docking appears to rationalize the experimentally determined regioselectivities. These calculations could also identify an active site residue (F33) as a putatively important interaction partner, a role that could explain the high degree of conservation of this residue during evolution. Overall, our combined experimental, structural and computational studies of this system provide snapshots into the evolution of enantioconvergence in StEH1 catalyzed epoxide hydrolysis.

    Keywords
    enantioselectivity; epoxide hydrolysis; evolutionary snapshots; laboratory evolution; protein engineering
    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-298675 (URN)10.1002/cbic.201600330 (DOI)000384425400004 ()27383542 (PubMedID)
    Funder
    Swedish Research CouncilEU, European Research Council, 306474Swedish National Infrastructure for Computing (SNIC), SNIC2015-16-12EU, FP7, Seventh Framework Programme, 283570
    Available from: 2016-07-06 Created: 2016-07-06 Last updated: 2017-11-28Bibliographically approved
    5. Epoxide Hydrolysis as a Model System for Understanding Flux Through a Branched Reaction Scheme
    Open this publication in new window or tab >>Epoxide Hydrolysis as a Model System for Understanding Flux Through a Branched Reaction Scheme
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    2018 (English)In: IUCrJ, ISSN 0972-6918, E-ISSN 2052-2525, Vol. 5, no 3, p. 269-282Article in journal (Refereed) Published
    Abstract [en]

    The epoxide hydrolase StEH1 catalyzes the hydrolysis of trans-methylstyrene oxide to 1-phenyl­propane-1,2-diol. The (S,S)-epoxide is exclusively transformed into the (1R,2S)-diol, while hydrolysis of the (R,R)-epoxide results in a mixture of product enantiomers. In order to understand the differences in the stereoconfigurations of the products, the reactions were studied kinetically during both the pre-steady-state and steady-state phases. A number of closely related StEH1 variants were analyzed in parallel, and the results were rationalized by structure–activity analysis using the available crystal structures of all tested enzyme variants. Finally, empirical valence-bond simulations were performed in order to provide additional insight into the observed kinetic behaviour and ratios of the diol product enantiomers. These combined data allow us to present a model for the flux through the catalyzed reactions. With the (R,R)-epoxide, ring opening may occur at either C atom and with similar energy barriers for hydrolysis, resulting in a mixture of diol enantiomer products. However, with the (S,S)-epoxide, although either epoxide C atom may react to form the covalent enzyme intermediate, only the pro-(R,S) alkylenzyme is amenable to subsequent hydrolysis. Previously contradictory observations from kinetics experiments as well as product ratios can therefore now be explained for this biocatalytically relevant enzyme.

    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-343750 (URN)10.1107/S2052252518003573 (DOI)000431151300004 ()29755743 (PubMedID)
    Funder
    Swedish Research CouncilEU, FP7, Seventh Framework Programme
    Available from: 2018-03-01 Created: 2018-03-01 Last updated: 2018-12-03Bibliographically approved
    6. Q Version 6, a comprehensive toolkit for empirical valence bond and related free energy calculations.
    Open this publication in new window or tab >>Q Version 6, a comprehensive toolkit for empirical valence bond and related free energy calculations.
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    (English)Manuscript (preprint) (Other academic)
    National Category
    Other Chemistry Topics
    Identifiers
    urn:nbn:se:uu:diva-325490 (URN)
    Available from: 2017-07-02 Created: 2017-07-02 Last updated: 2017-07-03
  • 14.
    Bauer, Paul
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Directed Evolution of ADH-A from Rhodococcus ruber DSM 445412014Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
  • 15.
    Bauer, Paul
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Janfalk Carlsson, Åsa
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Amrein, Beat A.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Kamerlin, S. C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Conformational Diversity and Enantioconvergence in Potato Epoxide Hydrolase 12016In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 14, no 24, p. 5639-5651Article in journal (Refereed)
    Abstract [en]

    Potato epoxide hydrolase 1 (StEH1) is a biocatalytically important enzyme that exhibits rich enantio-and regioselectivity in the hydrolysis of chiral epoxide substrates. In particular, StEH1 has been demonstrated to enantioconvergently hydrolyze racemic mixes of styrene oxide (SO) to yield (R)-1-phenylethanediol. This work combines computational, crystallographic and biochemical analyses to understand both the origins of the enantioconvergent behavior of the wild-type enzyme, as well as shifts in activities and substrate binding preferences in an engineered StEH1 variant, R-C1B1, which contains four active site substitutions (W106L, L109Y, V141K and I155V). Our calculations are able to reproduce both the enantio-and regioselectivities of StEH1, and demonstrate a clear link between different substrate binding modes and the corresponding selectivity, with the preferred binding modes being shifted between the wild-type enzyme and the R-C1B1 variant. Additionally, we demonstrate that the observed changes in selectivity and the corresponding enantioconvergent behavior are due to a combination of steric and electrostatic effects that modulate both the accessibility of the different carbon atoms to the nucleophilic side chain of D105, as well as the interactions between the substrate and protein amino acid side chains and active site water molecules. Being able to computationally predict such subtle effects for different substrate enantiomers, as well as to understand their origin and how they are affected by mutations, is an important advance towards the computational design of improved biocatalysts for enantioselective synthesis.

  • 16.
    Belfrage, Anna Karin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Drug Design and Discovery.
    Abdurakhmanov, Eldar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Åkerblom, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Drug Design and Discovery.
    Brandt, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Drug Design and Discovery.
    Alogheli, Hiba
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Drug Design and Discovery.
    Neyts, Johan
    Rega Institute, Department of Microbiology and Immunology, University of Leuven, B-3000 Leuven, Belgium.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Johansson, Anja
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Drug Design and Discovery.
    Pan-NS3 protease inhibitors of hepatitis C virus based on an R3-elongated pyrazinone scaffold2018In: European Journal of Medicinal Chemistry, ISSN 0223-5234, E-ISSN 1768-3254, Vol. 148, p. 453-464Article in journal (Refereed)
    Abstract [en]

    Herein, we present the design and synthesis of 2(1H)-pyrazinone based HCV NS3 protease inhibitors and show that elongated R-3 urea substituents were associated with increased inhibitory potencies over several NS3 protein variants. The inhibitors are believed to rely on beta-sheet mimicking hydrogen bonds which are similar over different genotypes and current drug resistant variants and correspond to the beta-sheet interactions of the natural peptide substrate. Inhibitor 36, for example, with a urea substituent including a cyclic imide showed balanced nanomolar inhibitory potencies against genotype la, both wild-type (K-i=30 nM) and R155K (K-i=2 nM), and genotype 3a (K-i=5 nM).

  • 17.
    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, p. 2603-2620Article 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.

  • 18.
    Berglin, Lennart
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Kjellander, Marcus
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Johansson, Gunnar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    In situ generation of iminodiacetic acid groups on nanoporous alumina for the reversible immobilization of enzymes and other biomolecules2014In: Biotechnology letters, ISSN 0141-5492, E-ISSN 1573-6776, Vol. 36, no 9, p. 1819-1825Article in journal (Refereed)
    Abstract [en]

    Nanoporous alumina membranes were silanized with aminopropylsilane and iminodiacetic acid (IDA) groups were generated in situ by reaction with iodoacetate. The membranes were mounted in standard filter holders, connected to a HPLC system and saturated with selected metal ions. Cu(II) allowed the capture of chicken muscle lactate dehydrogenase with such stability, repeatability and reproducibility that Michaelis-Menten kinetics could be studied. The IDA surface was stable for months and could be depleted and regenerated with metal ions multiple times without appreciable loss of capacity. The binding of lactate dehydrogenase influenced the backpressure to the extent that could be expected for a monolayer according to Poiseuilles law.

  • 19.
    Billinger, Erika
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Johansson, Gunnar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Kinetic studies of serine protease inhibitors in simple and rapid 'active barrier' model systems: Diffusion through an inhibitor barrier2018In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 546, p. 43-49Article in journal (Refereed)
    Abstract [en]

    A model based on gelatin for protease activity studies was designed. The model is also extended to study the efficiency of inhibitors in a separate protective layer covering the layer containing the target substrate. A good correlation between protease concentration and the size of erosion wells formed in a plain gelatin layer was observed. Similarly, increased concentration of inhibitors gave a systematic decrease in well area. Kinetic analyses of the two-layer model in a spectrophotometric plate reader with a fixed concentration of substrate in the bottom layer displayed a strict dependence of both inhibitor concentration and thickness of the top "protective" layer. An apparent, but weaker inhibition effect was also observed without inhibitors due to diffusional and erosion delay of enzyme transport to the substrate-containing layer.

  • 20.
    Blikstad, Cecilia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Dahlström, Kärthe M.
    Åbo Akademi.
    Salminen, Tiina A.
    Åbo Akademi.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Stereoselective oxidation of aryl-substituted vicinal diols into chiral α-hydroxy aldehydes by re-engineered propanediol oxidoreductase2013In: ACS Catalysis, ISSN 2155-5435, Vol. 3, no 12, p. 3016-3025Article in journal (Refereed)
    Abstract [en]

    α-Hydroxy aldehydes are chiral building blocks used in synthesis of natural products and synthetic drugs. One route to their production is by regioselective oxidation of vicinal diols and, in this work, we aimed to perform the oxidation of 3-phenyl-1,2-propanediol into the corresponding α‑hydroxy aldehyde applying enzyme catalysis. Propanediol oxidoreductase from E. coli efficiently catalyzes the stereoselective oxidation of S-1,2-propanediol into S-lactaldehyde. The enzyme, however, shows no detectable activity with aryl-substituted or other bulky alcohols. We conducted ISM-driven directed evolution on FucO and were able to isolate several mutants that were active with S-3-phenyl-1,2-propanediol. The most efficient variant displayed a kcat/KM of 40 s-1M-1 and the most enantioselective variant an E-value (S/R) of 80. Furthermore, other isolated variants showed up to 4400-fold increased activity with another bulky substrate, phenylacetaldehyde. The results with engineered propanediol oxidoreductases identified amino acids important for substrate selectivity and asymmetric synthesis of aryl-substituted α-hydroxy aldehydes. In conclusion, our study demonstrates the feasibility of tailoring the catalytic properties of propanediol oxidoreductase for biocatalytic properties.

  • 21.
    Blikstad, Cecilia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Dahlström, Käthe
    Salminen, Tiina
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Substrate scope and selectivity in offspring to an enzyme subjected to directed evolution2014In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 281, no 10, p. 2387-2398Article in journal (Refereed)
    Abstract [en]

    We have analyzed the effects of mutations inserted during directed evolution of a specialized enzyme, Escherichia coli S-1,2-propanediol oxidoreductase (FucO). The kinetic properties of evolved variants have been determined and the observed differences have been rationalized by modeling the tertiary structures of isolated variants and the wild-type enzyme. The native substrate, S-1,2-propanediol, as well as phenylacetaldehyde and 2S-3-phenylpropane-1,2-diol, which are new substrates accepted by isolated variants, were docked into the active sites. The study provides a comprehensive picture of how acquired catalytic properties have arisen via an intermediate generalist enzyme, which had acquired a single mutation (L259V) in the active site. Further mutagenesis of this generalist resulted in a new specialist catalyst. We have also been able to relate the native enzyme activities to the evolved ones and linked the differences to individual amino acid residues important for activity and selectivity. F254 plays a dual role in the enzyme function. First, mutation of F254 into an isoleucine weakens the interactions with the coenzyme thereby increasing its dissociation rate from the active site and resulting in a four-fold increase in turnover number with S-1,2-propanediol. Second, F254 is directly involved in binding of aryl-substituted substrates via π–π interactions. On the other hand, N151 is critical in determining the substrate scope since the side chain amide group stabilizes binding of 1,2-substituted diols and is apparently necessary for enzymatic activity with these substrates. Moreover, the side chain of N151 introduces steric hindrance, which prevents high activity with phenylacetaldehyde. Additionally, the hydroxyl group of T149 is required to maintain the catalytically important hydrogen bonding network.

  • 22.
    Blikstad, Cecilia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    High-throughput methods for identification of protein-protein interactions involving short linear motifs2015In: Cell Communication and Signaling, ISSN 1478-811X, E-ISSN 1478-811X, Vol. 13, article id 38Article, review/survey (Refereed)
    Abstract [en]

    Interactions between modular domains and short linear motifs (3-10 amino acids peptide stretches) are crucial for cell signaling. The motifs typically reside in the disordered regions of the proteome and the interactions are often transient, allowing for rapid changes in response to changing stimuli. The properties that make domain-motif interactions suitable for cell signaling also make them difficult to capture experimentally and they are therefore largely underrepresented in the known protein-protein interaction networks. Most of the knowledge on domain-motif interactions is derived from low-throughput studies, although there exist dedicated high-throughput methods for the identification of domain-motif interactions. The methods include arrays of peptides or proteins, display of peptides on phage or yeast, and yeast-two-hybrid experiments. We here provide a survey of scalable methods for domain-motif interaction profiling. These methods have frequently been applied to a limited number of ubiquitous domain families. It is now time to apply them to a broader set of peptide binding proteins, to provide a comprehensive picture of the linear motifs in the human proteome and to link them to their potential binding partners. Despite the plethora of methods, it is still a challenge for most approaches to identify interactions that rely on post-translational modification or context dependent or conditional interactions, suggesting directions for further method development.

  • 23.
    Calixto, Ana R.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Moreira, Catia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Pabis, Anna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Kötting, Carsten
    Ruhr Univ Bochum, Dept Biophys, D-44801 Bochum, Germany.
    Gerwert, Klaus
    Ruhr Univ Bochum, Dept Biophys, D-44801 Bochum, Germany.
    Rudack, Till
    Ruhr Univ Bochum, Dept Biophys, D-44801 Bochum, Germany.
    Kamerlin, Shina C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    GTP Hydrolysis Without an Active Site Base: A Unifying Mechanism for Ras and Related GTPases2019In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 141, no 27, p. 10684-10701Article in journal (Refereed)
    Abstract [en]

    GTP hydrolysis is a biologically crucial reaction, being involved in regulating almost all cellular processes. As a result, the enzymes that catalyze this reaction are among the most important drug targets. Despite their vital importance and decades of substantial research effort, the fundamental mechanism of enzyme-catalyzed GTP hydrolysis by GTPases remains highly controversial. Specifically, how do these regulatory proteins hydrolyze GTP without an obvious general base in the active site to activate the water molecule for nucleophilic attack? To answer this question, we perform empirical valence bond simulations of GTPase-catalyzed GTP hydrolysis, comparing solvent- and substrate-assisted pathways in three distinct GTPases, Ras, Rab, and the G(alpha i), subunit of a heterotrimeric G-protein, both in the presence and in the absence of the corresponding GTPase activating proteins. Our results demonstrate that a general base is not needed in the active site, as the preferred mechanism for GTP hydrolysis is a conserved solvent-assisted pathway. This pathway involves the rate-limiting nucleophilic attack of a water molecule, leading to a short-lived intermediate that tautomerizes to form H2PO4- and GDP as the final products. Our fundamental biochemical insight into the enzymatic regulation of GTP hydrolysis not only resolves a decades-old mechanistic controversy but also has high relevance for drug discovery efforts. That is, revisiting the role of oncogenic mutants with respect to our mechanistic findings would pave the way for a new starting point to discover drugs for (so far) "undruggable" GTPases like Ras.

  • 24.
    Cho, S. Ei
    et al.
    Stanford Univ, Biol, Stanford, CA USA.
    Roy, J.
    Stanford Univ, Biol, Stanford, CA USA.
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Cyert, M. S.
    Stanford Univ, Biol, Stanford, CA USA.
    Investigating the phospho-regulation of ER shaping protein RTN1A (Reticulon-1A) by the Calcineurin phosphatase.2017In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 28, no 26, p. 3727-3727Article in journal (Other academic)
  • 25.
    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, p. 4279-4293Article 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.

  • 26.
    Costeira-Paulo, Joana
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Gault, Joseph
    University of Oxford.
    Popova, Gergana
    Ladds, Marcus J G W
    van Leeuwen, Ingeborg M M
    Sarr, Médoune
    Olsson, Anders
    Lane, David P
    Laín, Sonia
    Marklund, Erik
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Landreh, Michael
    Lipids Shape the Electron Acceptor-Binding Site of the Peripheral Membrane Protein Dihydroorotate Dehydrogenase2018In: Cell Chemical Biology, ISSN 2451-9456, E-ISSN 2451-9448, Vol. 25, no 3, p. 309-317Article in journal (Refereed)
    Abstract [en]

    The interactions between proteins and biological membranes are important for drug development, but remain notoriously refractory to structural investigation. We combine non-denaturing mass spectrometry (MS) with molecular dynamics (MD) simulations to unravel the connections among co-factor, lipid, and inhibitor binding in the peripheral membrane protein dihydroorotate dehydrogenase (DHODH), a key anticancer target. Interrogation of intact DHODH complexes by MS reveals that phospholipids bind via their charged head groups at a limited number of sites, while binding of the inhibitor brequinar involves simultaneous association with detergent molecules. MD simulations show that lipids support flexible segments in the membrane-binding domain and position the inhibitor and electron acceptor-binding site away from the membrane surface, similar to the electron acceptor-binding site in respiratory chain complex I. By complementing MS with MD simulations, we demonstrate how a peripheral membrane protein uses lipids to modulate its structure in a similar manner as integral membrane proteins.

  • 27.
    Dahlström, Kathe M.
    et al.
    Abo Akad Univ, Struct Bioinformat Lab, Biochem, Turku, Finland..
    Blikstad, Cecilia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Salminen, Tiina A.
    Abo Akad Univ, Struct Bioinformat Lab, Biochem, Turku, Finland..
    Directed evolution on FucO - structural explanations for changes in substrate scope2015In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 24, p. 199-200Article in journal (Other academic)
  • 28.
    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, p. 223-240Conference 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.

  • 29.
    Davey, Norman E.
    et al.
    Univ Coll Dublin, Conway Inst Biomol & Biomed Sci, Dublin 4, Ireland..
    Seo, Moon-Hyeong
    Univ Toronto, Terrence Donnelly Ctr Cellular & Biomol Res, Toronto, ON M5S 3E1, Canada..
    Yadav, Vikash Kumar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Jeon, Jouhyun
    Univ Toronto, Terrence Donnelly Ctr Cellular & Biomol Res, Toronto, ON M5S 3E1, Canada..
    Nim, Satra
    Univ Toronto, Terrence Donnelly Ctr Cellular & Biomol Res, Toronto, ON M5S 3E1, Canada..
    Krystkowiak, Izabella
    Univ Coll Dublin, Conway Inst Biomol & Biomed Sci, Dublin 4, Ireland..
    Blikstad, Cecilia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Dong, Debbie
    Univ Toronto, Terrence Donnelly Ctr Cellular & Biomol Res, Toronto, ON M5S 3E1, Canada..
    Markova, Natalia
    Malvern Instruments Nord AB, Solna, Sweden..
    Kim, Philip M.
    Univ Toronto, Terrence Donnelly Ctr Cellular & Biomol Res, Toronto, ON M5S 3E1, Canada.;Univ Toronto, Dept Mol Genet, Toronto, ON M5S 1A1, Canada.;Univ Toronto, Dept Comp Sci, Toronto, ON M5S 1A1, Canada..
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Discovery of short linear motif-mediated interactions through phage display of intrinsically disordered regions of the human proteome2017In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 284, no 3, p. 485-498Article in journal (Refereed)
    Abstract [en]

    The intrinsically disordered regions of eukaryotic proteomes are enriched in short linear motifs (SLiMs), which are of crucial relevance for cellular signaling and protein regulation; many mediate interactions by providing binding sites for peptide-binding domains. The vast majority of SLiMs remain to be discovered highlighting the need for experimental methods for their large-scale identification. We present a novel proteomic peptide phage display (ProP-PD) library that displays peptides representing the disordered regions of the human proteome, allowing direct large-scale interrogation of most potential binding SLiMs in the proteome. The performance of the ProP-PD library was validated through selections against SLiM-binding bait domains with distinct folds and binding preferences. The vast majority of identified binding peptides contained sequences that matched the known SLiM-binding specificities of the bait proteins. For SHANK1 PDZ, we establish a novel consensus TxF motif for its non-C-terminal ligands. The binding peptides mostly represented novel target proteins, however, several previously validated protein-protein interactions (PPIs) were also discovered. We determined the affinities between the VHS domain of GGA1 and three identified ligands to 40-130 mu M through isothermal titration calorimetry, and confirmed interactions through coimmunoprecipitation using full-length proteins. Taken together, we outline a general pipeline for the design and construction of ProP-PD libraries and the analysis of ProP-PD-derived, SLiM-based PPIs. We demonstrated the methods potential to identify low affinity motif-mediated interactions for modular domains with distinct binding preferences. The approach is a highly useful complement to the current toolbox of methods for PPI discovery.

  • 30.
    Dobritzsch, Doreen
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Wang, Huaming
    Schneider, Gunter
    Yu, Shukun
    Structural and functional characterization of ochratoxinase, a novel mycotoxin-degrading enzyme2014In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 462, no 3, p. 441-452Article in journal (Refereed)
    Abstract [en]

    Ochratoxin, with ochratoxin A as the dominant form, is one of the five major mycotoxins most harmful to humans and animals. It is produced by Aspergillus and Penicillium species and occurs in a wide range of agricultural products. Detoxification of contaminated food is a challenging health issue. In the present paper we report the identification, characterization and crystal structure (at 2.2 angstrom) of a novel microbial ochratoxinase from Aspergillus niger. A putative amidase gene encoding a 480 amino acid polypeptide was cloned and homologously expressed in A. niger. The recombinant protein is N-terminally truncated, thermostable, has optimal activity at pH similar to 6 and 66 degrees C, and is more efficient in ochratoxin A hydrolysis than carboxypeptidase A and Y, the two previously known enzymes capable of degrading this mycotoxin. The subunit of the homo-octameric enzyme folds into a two-domain structure characteristic of a metal dependent amidohydrolase, with a twisted TIM (triosephosphateisomerase)-barrel and a smaller beta-sandwich domain. The active site contains an aspartate residue for acid base catalysis, and a carboxylated lysine and four histidine residues for binding of a binuclear metal centre.

  • 31. 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, p. 4838-4847Article 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.

  • 32. Dourado, Daniel F. A. R.
    et al.
    Fernandes, Pedro Alexandrino
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Ramos, Maria Joao
    Isomerization of Delta(5)-Androstene-3,17-dione into Delta(4)-Androstene-3, 17-dione Catalyzed by Human Glutathione Transferase A3-3: A Computational Study Identifies a Dual Role for Glutathione2014In: Journal of Physical Chemistry A, ISSN 1089-5639, E-ISSN 1520-5215, Vol. 118, no 31, p. 5790-5800Article in journal (Refereed)
    Abstract [en]

    Glutathione transferases (GSTs) are important enzymes in the metabolism of electrophilic xenobiotic and endobiotic toxic compounds. In addition, human GST A3-3 also catalyzes the double bond isomerization of Delta 5-androstene-3,17-dione (Delta(5)-AD) and Delta(5)-pregnene-3,20-dione (Delta(5)-PD), which are the immediate precursors of testosterone and progesterone. In fact, GST A3-3 is the most efficient human enzyme known to exist in the catalysis of these reactions. In this work, we have used density functional theory (DFT) calculations to propose a refined mechanism for the isomerization of Delta(5)-AD catalyzed by GST A3-3. In this mechanism the glutathione (GSH) thiol and Tyr9 catalyze the proton transfer from the Delta(5)-AD C4 atom to the Delta(5)-AD C6 atom, with a rate limiting activation energy of 15.8 kcal.mol(-1). GSH has a dual function, because it is also responsible for stabilizing the negative charge that is formed in the 03 atom of the enolate intermediate. The catalytic role of Tyr9 depends on significant conformational rearrangements of its side chain. Neither of these contributions to catalysis has been observed before. Residues Phe10, Leul11, Ala 208, and Ala 216 complete the list of the important catalytic residues. The mechanism detailed here is based on the GST A3-3:GSH:Delta(4)-AD crystal structure and is consistent with all available experimental data.

  • 33.
    Dourado, Daniel F. A. R.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Fernandes, Pedro Alexandrino
    Ramos, Maria Joao
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Mechanism of Glutathione Transferase P1-1-Catalyzed Activation of the Prodrug Canfosfamide (TLK286, TELCYTA)2013In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, no 45, p. 8069-8078Article in journal (Refereed)
    Abstract [en]

    Canfosfamide (TLK286, TELCYTA) is a prodrug that upon activation by glutathione transferase P1-1 (GST P1-1) yields an anticancer alkylating agent and a glutathione derivative. The rationale underlying the use of TLK286 in chemotherapy is that tumor cells overexpressing GST P1-1 will be locally exposed to the released alkylating agent with limited collateral toxicity to the surrounding normal tissues. TLK286 has demonstrated clinical effects in phase II and III clinical trials for the treatment of malignancies, such as ovarian cancer, nonsmall cell lung cancer, and breast cancer, as a single agent and in combination with other chemotherapeutic agents. In spite of these promising results, the detailed mechanism of GST P1-1 activation of the prodrug has not been elucidated. Here, we propose a mechanism for the TLK286 activation by GST P1-1 on the basis of density functional theory (DFT) and on potential of mean force (PMF) calculations. A catalytic water molecule is instrumental to the activation by forming a network of intermolecular interactions between the active-site Tyr7 hydroxyl and the sulfone and COO- groups of TLK286. The results obtained are consistent with the available experimental kinetic data and provide an atomistic understanding of the TLK286 activation mechanism.

  • 34.
    Drevinskas, Tomas
    et al.
    Vytautas Magnus Univ, Fac Nat Sci, Instrumental Anal Open Access Ctr, Vileikos Str 8, LT-44404 Kaunas, Lithuania;Vytautas Magnus Univ, Fac Informat, Dept Syst Anal, Vileikos Str 8, LT-44404 Kaunas, Lithuania.
    Maruska, Audrius
    Vytautas Magnus Univ, Fac Nat Sci, Instrumental Anal Open Access Ctr, Vileikos Str 8, LT-44404 Kaunas, Lithuania.
    Telksnys, Laimutis
    Vytautas Magnus Univ, Fac Informat, Dept Syst Anal, Vileikos Str 8, LT-44404 Kaunas, Lithuania;Vilnius Univ, Inst Data Sci & Digital Technol, Gostauto 12, LT-01108 Vilnius, Lithuania.
    Hjertén, Stellan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Stankevicius, Mantas
    Vytautas Magnus Univ, Fac Nat Sci, Instrumental Anal Open Access Ctr, Vileikos Str 8, LT-44404 Kaunas, Lithuania.
    Lelesius, Raimundas
    Lithuanian Univ Hlth Sci, Vet Acad, Dept Vet Pathobiol, Tilzes Str 18, LT-47181 Kaunas, Lithuania;Lithuanian Univ Hlth Sci, Vet Acad, Inst Microbiol & Virol, Tilzes Str 18, LT-47181 Kaunas, Lithuania.
    Mickiene, Ruta
    Vytautas Magnus Univ, Fac Nat Sci, Instrumental Anal Open Access Ctr, Vileikos Str 8, LT-44404 Kaunas, Lithuania.
    Karpovaite, Agneta
    Lithuanian Univ Hlth Sci, Vet Acad, Dept Vet Pathobiol, Tilzes Str 18, LT-47181 Kaunas, Lithuania.
    Salomskas, Algirdas
    Lithuanian Univ Hlth Sci, Vet Acad, Dept Vet Pathobiol, Tilzes Str 18, LT-47181 Kaunas, Lithuania.
    Tiso, Nicola
    Vytautas Magnus Univ, Fac Nat Sci, Instrumental Anal Open Access Ctr, Vileikos Str 8, LT-44404 Kaunas, Lithuania.
    Ragazinskiene, Ona
    Vytautas Magnus Univ, Kaunas Bot Garden, Sect Med Plants, ZE Zilibero Str 6, LT-46324 Kaunas, Lithuania.
    Chromatographic Data Segmentation Method: A Hybrid Analytical Approach for the Investigation of Antiviral Substances in Medicinal Plant Extracts2019In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 91, no 1, p. 1080-1088Article in journal (Refereed)
    Abstract [en]

    The methodology described in this article will significantly reduce the time required for understanding the relations between chromatographic data and bioactivity assays. The methodology is a hybrid of hypothesis-based and data driven scientific approaches. In this work, a novel chromatographic data segmentation method is proposed, which demonstrates the capability of finding what volatile substances are responsible for antiviral and cytotoxic effects in the medicinal plant extracts. Up until now, the full potential of the separation methods has not been exploited in the life sciences. This was due to the lack of data ordering methods capable of adequately preparing the chromatographic information. Furthermore, the data analysis methods suffer from multidimensionality, requiring a large number of investigated data points. A new method is described for processing any chromatographic information into a vector. The obtained vectors of highly complex and different origin samples can be compared mathematically. The proposed method, efficient with relatively small sized data sets, does not suffer from multidimensionality. In this novel analytical approach, the samples did not need fractionation and purification, which is typically used in hypothesis-based scientific research. All investigations were performed using crude extracts possessing hundreds of phyto-substances. The antiviral properties of medicinal plant extracts were investigated using gas chromatography-mass spectrometry, antiviral tests, and proposed data analysis methods. The findings suggested that (i) beta-cis-caryophyllene, linalool, and eucalyptol possess antiviral activity, while (ii) thujones do not, and (iii) alpha-thujone, beta-thujone, cis-p-menthan-3-one, and estragole show cytotoxic effects.

  • 35.
    Egea-Jimenez, Antonio Luis
    et al.
    Aix Marseille Univ, Inst Paoli Calmettes, INSERM, CRCM,U1068,CNRS UMR7258, F-13009 Marseille, France; Katholieke Univ Leuven, Dept Human Genet, ON1 Herestr 49,Box 602, B-3000 Leuven, Belgium.
    Gallardo, Rodrigo
    Katholieke Univ Leuven, Dept Human Genet, ON1 Herestr 49,Box 602, B-3000 Leuven, Belgium;Katholieke Univ Leuven, Dept Mol Cellular & Mol Med, VIB, VIB Switch Lab, B-3000 Leuven, Belgium.
    Garcia-Pino, Abel
    Vrije Univ Brussel, Struct Biol Brussels, Dept Biotechnol DBIT, Pl Laan 2, B-1050 Brussels, Belgium; VIB, Mol Recognit Unit, Struct Biol Res Ctr, Pl Laan 2, B-1050 Brussels, Belgium; Univ Libre Bruxelles, Biol Struct & Biophys, CP300,Rue Prof Jeener & Brachet 12, B-6041 Gosselies, Belgium.
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Katholieke Univ Leuven, Dept Human Genet, ON1 Herestr 49,Box 602, B-3000 Leuven, Belgium.
    Wawrzyniak, Anna Maria
    Katholieke Univ Leuven, Dept Human Genet, ON1 Herestr 49,Box 602, B-3000 Leuven, Belgium.
    Kashyap, Rudra
    Aix Marseille Univ, Inst Paoli Calmettes, INSERM, CRCM,U1068,CNRS UMR7258, F-13009 Marseille, France; Katholieke Univ Leuven, Dept Human Genet, ON1 Herestr 49,Box 602, B-3000 Leuven, Belgium.
    Loris, Remy
    Vrije Univ Brussel, Struct Biol Brussels, Dept Biotechnol DBIT, Pl Laan 2, B-1050 Brussels, Belgium; VIB, Mol Recognit Unit, Struct Biol Res Ctr, Pl Laan 2, B-1050 Brussels, Belgium.
    Schymkowitz, Joost
    Katholieke Univ Leuven, Dept Mol Cellular & Mol Med, VIB, VIB Switch Lab, B-3000 Leuven, Belgium.
    Rousseau, Frederic
    Katholieke Univ Leuven, Dept Mol Cellular & Mol Med, VIB, VIB Switch Lab, B-3000 Leuven, Belgium.
    Zimmermann, Pascale
    Aix Marseille Univ, Inst Paoli Calmettes, INSERM, CRCM,U1068,CNRS UMR7258, F-13009 Marseille, France; Katholieke Univ Leuven, Dept Human Genet, ON1 Herestr 49,Box 602, B-3000 Leuven, Belgium.
    Frizzled 7 and PIP2 binding by syntenin PDZ2 domain supports Frizzled 7 trafficking and signalling2016In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 7, article id 12101Article in journal (Refereed)
    Abstract [en]

    PDZ domain-containing proteins work as intracellular scaffolds to control spatio-temporal aspects of cell signalling. This function is supported by the ability of their PDZ domains to bind other proteins such as receptors, but also phosphoinositide lipids important for membrane trafficking. Here we report a crystal structure of the syntenin PDZ tandem in complex with the carboxy-terminal fragment of Frizzled 7 and phosphatidylinositol 4,5-bisphosphate (PIP2). The crystal structure reveals a tripartite interaction formed via the second PDZ domain of syntenin. Biophysical and biochemical experiments establish co-operative binding of the tripartite complex and identify residues crucial for membrane PIP2-specific recognition. Experiments with cells support the importance of the syntenin-PIP2 interaction for plasma membrane targeting of Frizzled 7 and c-jun phosphorylation. This study contributes to our understanding of the biology of PDZ proteins as key players in membrane compartmentalization and dynamics.

  • 36.
    Ehrenberg, Angelica
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Novel Procedures for Identification and Characterization of Viral Proteases Inhibitors2014Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Viral proteases are often considered to be attractive drug targets because of their crucial function in the viral replication machinery. In order to increase our knowledge of these important targets and to contribute to the discovery and development of new antiviral drugs, the proteases from hepatitis C virus (HCV) and human cytomegalovirus (HCMV) have been produced and their interactions with inhibitors and fragments have been characterized, using enzyme inhibition and SPR biosensor based interaction assay.

    The structure activity relationships and the resistance profiles of a series of HCV NS3 protease inhibitors based on either P2 proline or phenylglycine residues were analyzed using wild type genotype 1a and the major resistant variants A156T and D168V. The observed susceptibility to substitutions associated with these resistance variants was concluded to depend on the P2 and the P1 residue, and not only on the P2 residue as previously had been suggested. In order to be able to evaluate how the potency of inhibitors is affected by genetic variation, their effect was evaluated on wild type NS3 from genotype 1a, 1b and 3a as well as on the resistant variant R155K from genotype 1a. To enable a comparison of the inhibitory effect on the enzyme variants, the compounds were analyzed under conditions optimized for each variant. VX-950 was found to be the least susceptible compound to resistance and genetic variation. A more detailed analysis showed that the kinetic and mechanistic features of the inhibitors were significantly different for the different genotypes. The reversible non covalent macrocyclic inhibitor ITMN 191 was revealed to have favorable kinetics for all three genotypes. This is an advantage for the design of broad spectrum drugs.

    A fragment based procedure for identifying and validating novel scaffolds for inhibitors of HCMV protease was established. It identified fragments that may serve as starting points for the discovery of effective inhibitors against this challenging target.  

    The procedures developed for the evaluation and identification of novel HCV NS3 and HCMV protease inhibitors have contributed to a deeper understanding of protease-inhibitor interactions that is expected to have an impact on the design of novel antiviral drugs. 

    List of papers
    1. Structure-activity relationships of HCV NS3 protease inhibitors evaluated on the drug-resistant variants A156T and D168V
    Open this publication in new window or tab >>Structure-activity relationships of HCV NS3 protease inhibitors evaluated on the drug-resistant variants A156T and D168V
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    2010 (English)In: Antiviral Therapy, ISSN 1359-6535, E-ISSN 2040-2058, Vol. 15, no 6, p. 841-852Article in journal (Refereed) Published
    Abstract [en]

    BACKGROUND: HCV infections are a serious threat to public health. An important drug target is the NS3 protease, for which several inhibitors are in clinical trials. Because of the high mutation rate of the virus, resistance against any HCV-specific drug is likely to become a substantial problem. Structure-activity data for the major resistant variants are therefore needed to guide future designs of protease inhibitors. METHODS: The inhibitory potency of tripeptide NS3 protease inhibitors, with either a P2 proline or phenylglycine, in combination with different P3 and P1-P1' groups, was assessed in enzyme activity assays using the full-length NS3 protein with known resistance-conferring substitutions A156T or D168V. The results obtained from these variants were compared with the inhibition of the wild-type enzyme. Molecular modelling was used to rationalize the biochemical results. RESULTS: Inhibitors combining the P2 proline and P1 (1R,2S)-1-amino-2-vinylcyclopropyl-carboxylic acid (vinylACCA) lost much of their potency on the resistant variants. Exchange of the P2 proline for phenylglycine yielded inhibitors that were equipotent on the wild-type and on the A156T and D168V variants. The same result was obtained from the combination of either the P2 residue with a norvaline or an aromatic scaffold in the P1 position. CONCLUSIONS: The combination of a substituted P2 proline and P1 vinylACCA appears to be the main problem behind the observed resistance. Molecular modelling suggests an enforced change in binding conformation for the P2 proline-based inhibitors, whereas the phenylglycine-based inhibitors retained their wild-type binding conformation in the substituted forms of the enzyme.

    National Category
    Pharmaceutical Sciences Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-131962 (URN)10.3851/IMP1655 (DOI)000282390300004 ()20834096 (PubMedID)
    Available from: 2010-10-12 Created: 2010-10-12 Last updated: 2018-01-12Bibliographically approved
    2. Accounting for strain variations and resistance mutations in the characterization of hepatitis C NS3 protease inhibitors
    Open this publication in new window or tab >>Accounting for strain variations and resistance mutations in the characterization of hepatitis C NS3 protease inhibitors
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    2014 (English)In: Journal of enzyme inhibition and medicinal chemistry (Print), ISSN 1475-6366, E-ISSN 1475-6374, Vol. 29, no 6, p. 868-876Article in journal (Refereed) Published
    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.

    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-193252 (URN)10.3109/14756366.2013.864651 (DOI)000345518000013 ()24517372 (PubMedID)
    Available from: 2013-01-29 Created: 2013-01-29 Last updated: 2017-12-06Bibliographically approved
    3. Identification of Weak Points of Hepatitis C Virus NS3 Protease Inhibitors Using Surface Plasmon Resonance Biosensor-Based Interaction Kinetic Analysis and Genetic Variants
    Open this publication in new window or tab >>Identification of Weak Points of Hepatitis C Virus NS3 Protease Inhibitors Using Surface Plasmon Resonance Biosensor-Based Interaction Kinetic Analysis and Genetic Variants
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    2014 (English)In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 57, no 5, p. 1802-1811Article in journal (Other academic) Published
    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:uu:diva-193251 (URN)10.1021/jm401690f (DOI)000333005800012 ()
    Available from: 2013-01-29 Created: 2013-01-29 Last updated: 2017-12-06Bibliographically approved
    4. Challenges in the discovery of fragment targeting human cytomegalovirus protease
    Open this publication in new window or tab >>Challenges in the discovery of fragment targeting human cytomegalovirus protease
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    (English)Manuscript (preprint) (Other academic)
    Keywords
    CMV, cytomagalovirus, protease, SPR, biosensor, fragments-based drug discovery, screening, inhibitor
    National Category
    Natural Sciences
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-215703 (URN)
    Available from: 2014-01-15 Created: 2014-01-15 Last updated: 2014-02-10
  • 37.
    Eklund, Sandra
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Lindås, Ann-Christin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Hamnevik, Emil
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Tomkinson, Birgitta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Exploring the active site of tripeptidyl-peptidase II through studies of pH dependence of reaction kinetics2012In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1824, no 4, p. 561-570Article in journal (Refereed)
    Abstract [en]

    Tripeptidyl-peptidase II (TPP II) is a subtilisin-like serine protease which forms a large enzyme complex (> 4 MDa). It is considered a potential drug target due to its involvement in specific physiological processes. However, information is scarce concerning the kinetic characteristics of TPP II and its active site features, which are important for design of efficient inhibitors. To amend this, we probed the active site by determining the pH dependence of TPP II catalysis. Access to pure enzyme is a prerequisite for kinetic investigations and herein we introduce the first efficient purification system for heterologously expressed mammalian TPP II. The pH dependence of kinetic parameters for hydrolysis of two different chromogenic substrates, Ala-Ala-Phe-pNA and Ala-Ala-Ala-pNA, was determined for murine, human and Drosophila melanogaster TPP II as well as mutant variants thereof. The investigation demonstrated that TPP II, in contrast to subtilisin, has a bell-shaped pH dependence of kcatapp/KM probably due to deprotonation of the N-terminal amino group of the substrate at higher pH. Since both the KM and kcatapp are lower for cleavage of AAA-pNA than for AAF-pNA we propose that the former can bind non-productively to the active site of the enzyme, a phenomenon previously observed with some substrates for subtilisin. Two mutant variants, H267A and D387G, showed bell-shaped pH-dependence of kcatapp, possibly due to an impaired protonation of the leaving group. This work reveals previously unknown differences between TPP II orthologues and subtilisin as well as features that might be conserved within the entire family of subtilisin-like serine peptidases.

  • 38.
    Ekman, Pia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Eller, Marika
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Ragnarsson, Ulf
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Engström, Lorentz
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Two methods to avoid the effect of endogenous inhibitors during the assay of protein kinase C activity in tissue extracts.1992In: Preparative Biochemistry, ISSN 0032-7484, Vol. 22, no 2, p. 165-175Article in journal (Refereed)
    Abstract [en]

    Using H1 as substrate the protein kinase C activity of rat liver cell sap was increased about fourfold by treatment with DEAE-cellulose at pH 7.5 at an intermediate ionic strength due to removal of protein inhibitors. The activity of cell sap from rat spleen, brain or muscle was about doubled by the same treatment. In contrast, when a specific synthetic peptide substrate was used the corresponding increase of enzyme activity was not obtained when the inhibitors were removed. This shows that this type of substrates should be preferred for reliable assays of protein kinase C in crude extracts. The possible role of the protein inhibitors for the substrate specificity of protein kinase C is briefly discussed.

  • 39.
    Eller, Marika
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Järv, Jaak
    Tartu universitet.
    Toomik, Reet
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ragnarsson, Ulf
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Ekman, Pia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Engström, Lorentz
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Peptide fragments of myelin basic protein as substrates of protein kinase C.1992In: Biochemistry International, ISSN 0158-5231, Vol. 27, no 4, p. 625-631Article in journal (Refereed)
    Abstract [en]

    A set of peptides derived from myelin basic protein was synthesized and the kinetics of their phosphorylation by protein kinase C was studied. The replacement or the removal of the N-terminal Gln had no effect on the activity of the parent peptide. The removal of the following Lys or Arg led to a systematic decrease in substrate activity. The modifications in the C-terminal part of the peptide had a weaker influence on the parameters Vmax and KM than those in the N-terminal. The rather regular dependence of the activity of substrates upon their structure does not allow the strict definition of a minimum substrate for protein kinase C.

  • 40.
    Eller, Marika
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Järv, Jaak
    Tartu universitet.
    Toomik, Reet
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Ragnarsson, Ulf
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Ekman, Pia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Engström, Lorentz
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Substrate specificity of protein kinase C studied with peptides containing D-amino acid residues.1993In: Journal of Biochemistry, ISSN 0021-924X, Vol. 114, no 2, p. 177-180Article in journal (Refereed)
    Abstract [en]

    A set of stereoisomeric nonapeptides KRPSQRAKY with one, two, or all L-amino acid residues replaced by the corresponding D-amino acids, and two analogs with L- and D-threonine instead of serine, were synthesized and tested as substrates for protein kinase C. All of the peptides were phosphorylated by the enzyme. The maximal rate of the reaction with the all-D peptide was more than one order of magnitude lower than that for all-L peptide with serine. The same applied to the peptides with D-Ser or with D-Arg in position +2 with respect to Ser. The Km values for the peptides containing one D-amino acid were close to that for the prototype peptide (53 microM). On the other hand, when two or more D-amino acids were present, the Km value increased considerably. Replacement of serine by threonine also reduced the phosphorylation rate and increased the Km values. One can conclude that the stereospecificity of protein kinase C is much less pronounced than that of protein kinase A, which is in agreement with the less clearly pronounced substrate specificity of the former enzyme.

  • 41.
    Eller, Marika
    et al.
    Tartu universitet.
    Sepp, A
    Tartu universitet.
    Toomik, Reet
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ekman, Pia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Järv, Jaak
    Tartu universitet.
    Ragnarsson, Ulf
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Engström, Lorentz
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Studies on the substrate specificity of cAMP-dependent protein kinase using diastereomeric peptides1991In: Biochemistry International, ISSN 0158-5231, Vol. 25, no 3, p. 453-460Article in journal (Refereed)
    Abstract [en]

    A set of six different diastereomeric hexapeptides RRASVA, each with a D-amino acid residue successively in the six positions, was synthesized and tested as substrates of protein kinase A. It was found that the peptide with D-Ser was neither a substrate, nor an inhibitor of the enzyme. The other five peptides were active as substrates with slightly lower kcat values than that of the all-L amino acid peptide. However, the apparent Km values increased by one to two orders of magnitude, especially when the second arginine or the alanine residue preceding the serine was substituted. The results are discussed.

  • 42.
    Erik, Gioeli
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Utbytesoptimering inom läkemedelsproduktion: Identifiering av kassationsorsaker inom AstraZenecas packningsprocess i Gärtuna2017Independent thesis Advanced level (professional degree), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    The project aimed to deliver recommendations of how to optimize the yield of thepackaging process of tablets and capsules within AstraZeneca's facility in Gärtuna(Södertälje), Sweden. The project scope included nine packaging lines of blisterproducts with highly similar process steps. The crucial part of the puzzle was toidentify cassation sources within these production lines, which was achieved by thecombined methology of qualitative interviews, quantitative surveys and statisticalprocess control applied to logged production data.The following cassation sources were identified:- Cassation as a side effect of start-up controls within an order- Safety cassation of tablets when the blister machine stops- In many cases, the safety cassation also occours when the cartoner stops- A cut-in-half tablet causing cassation of multiple tablets in the rejection steps- Leftover product on the packaging line when the order is finished- Target conflict between high productivity of the production line and high yield withinthe order- Difficulty optimizing machines settings for small order sizes, leading to higher stopfrequency and therefore more safety cassation of tablets, as well as a higher risk thattablets are sorted out in control stepsWhich led to these recommendations:- Optimizing the yield as a target value for process optimization- Log the yield for all the process steps within the packaging line- Consider the possibility of reintroducing rejected blisters containing approvedtablets- Clear prioritization of the target conflict between high productivity and high yield- Analyze if there are any time-consuming steps conflicting with high yield within theprocess of closing an order which are not required to be performed at that particularprocess stepAs well as future work:- Get to the bottom of the correlation between low yield and small order sizes- Investigate further which materials are prone to cause machine stops within thecartoner- Dig deeper into the problem of cut-in-half tablets existing pre-packaging-process

  • 43. Filonova, Lada
    et al.
    Kallas, Åsa
    Greffe, Lionel
    Johansson, Gunnar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Teeri, Tuula
    Daniel, Geoffrey
    Analysis of the Surfaces of Wood Tissues and Pulp Fibers Using Carbohydrate-Binding Modules Specific for Crystalline Cellulose and Mannan2007In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 8, p. 91-97Article in journal (Refereed)
  • 44.
    Gabelica, Valérie
    et al.
    University Bordeaux, INSERM, CNRS, Laboratoire Acides Nucléiques Régulations Naturelle et Artificielle (ARNA, U1212, UMR5320), IECB, Pessac, France.
    Marklund, Erik
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Fundamentals of ion mobility spectrometry2018In: Current opinion in chemical biology, ISSN 1367-5931, E-ISSN 1879-0402, Vol. 42, p. 51-59, article id S1367-5931(17)30122-9Article, review/survey (Refereed)
    Abstract [en]

    Fundamental questions in ion mobility spectrometry have practical implications for analytical applications in general, and omics in particular, in three respects. (1) Understanding how ion mobility and collision cross section values depend on the collision gas, on the electric field and on temperature is crucial to ascertain their transferability across instrumental platforms. (2) Predicting collision cross section values for new analytes is necessary to exploit the full potential of ion mobility in discovery workflows. (3) Finally, understanding the fate of ion structures in the gas phase is essential to infer meaningful information on solution structures based on gas-phase ion mobility measurements. We review here the most recent advances in ion mobility fundamentals, relevant to these three aspects.

  • 45. Ganesan, Ashok
    et al.
    Debulpaep, Maja
    Wilkinson, Hannah
    Van Durme, Joost
    De Baets, Greet
    Jonckheere, Wim
    Ramakers, Meine
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Zimmermann, Pascale
    Van Eldere, Johan
    Schymkowitz, Joost
    Rousseau, Frederic
    Selectivity of Aggregation-Determining Interactions2015In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 427, no 2, p. 236-247Article in journal (Refereed)
    Abstract [en]

    Protein aggregation is sequence specific, favoring self-assembly over cross-seeding with non-homologous sequences. Still, as the majority of proteins in a proteome are aggregation prone, the high level of homogeneity of protein inclusions in vivo both during recombinant overexpression and in disease remains surprising. To investigate the selectivity of protein aggregation in a proteomic context, we here compared the selectivity of aggregation-determined interactions with antibody binding. To that purpose, we synthesized biotin-labeled peptides, corresponding to aggregation-determining sequences of the bacterial protein β-galactosidase and two human disease biomarkers: C-reactive protein and prostate-specific antigen. We analyzed the selectivity of their interactions in Escherichiacoli lysate, human serum and human seminal plasma, respectively, using a Western blot-like approach in which the aggregating peptides replace the conventional antibody. We observed specific peptide accumulation in the same bands detected by antibody staining. Combined spectroscopic and mutagenic studies confirmed accumulation resulted from binding of the peptide on the identical sequence of the immobilized target protein. Further, we analyzed the sequence redundancy of aggregating sequences and found that about 90% of them are unique within their proteome. As a result, the combined specificity and low sequence redundancy of aggregating sequences therefore contribute to the observed homogeneity of protein aggregation in vivo. This suggests that these intrinsic proteomic properties naturally compartmentalize aggregation events in sequence space. In the event of physiological stress, this might benefit the ability of cells to respond to proteostatic stress by allowing chaperones to focus on specific aggregation events rather than having to face systemic proteostatic failure.

  • 46. Garrido-Urbani, Sarah
    et al.
    Garg, Pankaj
    Ghossoub, Rania
    Arnold, Roland
    Lembo, Frédérique
    Sundell, Gustav N
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Kim, Philip M
    Lopez, Marc
    Zimmermann, Pascale
    Sidhu, Sachdev S
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Proteomic peptide phage display uncovers novel interactions of the PDZ1-2 supramodule of syntenin2016In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 590, no 1, p. 3-12Article in journal (Refereed)
    Abstract [en]

    Syntenin has crucial roles in cell adhesion, cell migration and synaptic transmission. Its closely linked postsynaptic density-95, discs large 1, zonula occludens-1 (PDZ) domains typically interact with C-terminal ligands. We profile syntenin PDZ1-2 through proteomic peptide phage display (ProP-PD) using a library that displays C-terminal regions of the human proteome. The protein recognizes a broad range of peptides, with a preference for hydrophobic motifs and has a tendency to recognize cryptic internal ligands. We validate the interaction with nectin-1 through orthogonal assays. The study demonstrates the power of ProP-PD as a complementary approach to uncover interactions of potential biological relevance.

  • 47.
    Gault, Joseph
    et al.
    University of Oxford, United Kingdom.
    Lianoudaki, Danai
    Kaldmäe, Margit
    Kronqvist, Nina
    Rising, Anna
    Johansson, Jan
    Lohkamp, Bernhard
    Laín, Sonia
    Allison, Timothy M.
    University of Canterbury, New Zealand.
    Lane, David P.
    Marklund, Erik
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Landreh, Michael
    Mass Spectrometry Reveals the Direct Action of a Chemical Chaperone2018In: Journal of Physical Chemistry Letters, ISSN 1948-7185, E-ISSN 1948-7185, Vol. 9, no 14, p. 4082-4086Article in journal (Refereed)
    Abstract [en]

    Despite their fundamental biological importance and therapeutic potential, the interactions between chemical chaperones and proteins remain difficult to capture due to their transient and nonspecific nature. Using a simple mass spectrometric assay, we are able to follow the interactions between proteins and the chemical chaperone trimethylamine-N-oxide (TMAO). In this manner, we directly observe that the counteraction of TMAO and the denaturant urea is driven by the exclusion of TMAO from the protein surface, whereas the surfactant lauryl dimethylamine-N-oxide cannot be displaced. Our results clearly demonstrate a direct chaperoning mechanism for TMAO, corroborating extensive computational studies, and pave the way for the use of nondenaturing mass spectrometry and related techniques to study chemical chaperones in molecular detail.

  • 48.
    Ge, Changrong P
    et al.
    Karolinska Inst, Sect Med Inflammat Res, Dept Med Biochem & Biophys, Stockholm, Sweden..
    Tong, Dongmei R
    Karolinska Inst, Sect Med Inflammat Res, Dept Med Biochem & Biophys, Stockholm, Sweden.;Southern Med Univ, Dept Pathophysiol, Key Lab Shock & Microcirculat Res Guangdong, Guangzhou, Guangdong, Peoples R China..
    Liang, Bibo T
    Karolinska Inst, Sect Med Inflammat Res, Dept Med Biochem & Biophys, Stockholm, Sweden.;Southern Med Univ, Dept Pathophysiol, Key Lab Shock & Microcirculat Res Guangdong, Guangzhou, Guangdong, Peoples R China..
    Lönnblom, Erik S
    Karolinska Inst, Sect Med Inflammat Res, Dept Med Biochem & Biophys, Stockholm, Sweden..
    Schneider, Nadine K
    Goethe Univ, Fraunhofer Inst Mol Biol & Appl Ecol IME, Project Grp Translat Med & Pharmacol, Frankfurt, Germany.;Goethe Univ, Div Rheumatol, Univ Hosp Frankfurt, Frankfurt, Germany..
    Hagert, Cecilia U
    Univ Turku, Medicity Res Lab, Turku, Finland.;Natl Doctoral Programme Informat & Struct Biol, Turku, Finland..
    Viljanen, Johan V.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Ayoglu, Burcu R
    KTH Royal Inst Technol, Sch Biotechnol, Sci Life Lab, Affin Prote, Stockholm, Sweden..
    Stawikowska, Roma T
    Florida Atlantic Univ, Dept Chem & Biochem, Jupiter, FL USA..
    Nilsson, Peter C
    KTH Royal Inst Technol, Sch Biotechnol, Sci Life Lab, Affin Prote, Stockholm, Sweden..
    Fields, Gregg B
    Florida Atlantic Univ, Dept Chem & Biochem, Jupiter, FL USA..
    Skogh, Thomas A
    Linkoping Univ, Dept Rheumatol, Dept Clin & Expt Med, Linkoping, Sweden..
    Kastbom, Alf R
    Linkoping Univ, Dept Rheumatol, Dept Clin & Expt Med, Linkoping, Sweden..
    Kihlberg, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Burkhardt, Harald T
    Goethe Univ, Fraunhofer Inst Mol Biol & Appl Ecol IME, Project Grp Translat Med & Pharmacol, Frankfurt, Germany.;Goethe Univ, Div Rheumatol, Univ Hosp Frankfurt, Frankfurt, Germany..
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Holmdahl, Rikard K
    Karolinska Inst, Sect Med Inflammat Res, Dept Med Biochem & Biophys, Stockholm, Sweden.;Univ Turku, Medicity Res Lab, Turku, Finland.;Natl Doctoral Programme Informat & Struct Biol, Turku, Finland.;Southern Med Univ, Ctr Med Immunopharmacol Res, Guangzhou, Guangdong, Peoples R China..
    Anti-citrullinated protein antibodies cause arthritis by cross-reactivity to joint cartilage2017In: JCI INSIGHT, ISSN 2379-3708, Vol. 2, no 13, article id e93688Article in journal (Refereed)
    Abstract [en]

    Today, it is known that autoimmune diseases start a long time before clinical symptoms appear. Anti-citrullinated protein antibodies (ACPAs) appear many years before the clinical onset of rheumatoid arthritis (RA). However, it is still unclear if and how ACPAs are arthritogenic. To better understand the molecular basis of pathogenicity of ACPAs, we investigated autoantibodies reactive against the C1 epitope of collagen type II (CII) and its citrullinated variants. We found that these antibodies are commonly occurring in RA. A mAb (ACC1) against citrullinated C1 was found to cross-react with several noncitrullinated epitopes on native CII, causing proteoglycan depletion of cartilage and severe arthritis in mice. Structural studies by X-ray crystallography showed that such recognition is governed by a shared structural motif "RG-TG" within all the epitopes, including electrostatic potential-controlled citrulline specificity. Overall, we have demonstrated a molecular mechanism that explains how ACPAs trigger arthritis.

  • 49.
    Ge, Changrong
    et al.
    Karolinska Inst, Stockholm, Sweden.
    Xu, Bingze
    Karolinska Inst, Stockholm, Sweden.
    Liang, Bibo
    Karolinska Inst, Stockholm, Sweden;Southern Med Univ, Guangzhou, Guangdong, Peoples R China.
    Lönnblom, Erik
    Karolinska Inst, Stockholm, Sweden.
    Lundström, Susanna L.
    Karolinska Inst, Stockholm, Sweden.
    Zubarev, Roman A.
    Karolinska Inst, Stockholm, Sweden.
    Ayoglu, Burcu
    KTH Royal Inst Technol, Stockholm, Sweden.
    Nilsson, Peter
    KTH Royal Inst Technol, Stockholm, Sweden.
    Skogh, Thomas
    Linkoping Univ, Linkoping, Sweden.
    Kastbom, Alf
    Linkoping Univ, Linkoping, Sweden.
    Malmström, Vivianne
    Karolinska Inst, Stockholm, Sweden;Karolinska Univ Hosp, Stockholm, Sweden.
    Klareskog, Lars
    Karolinska Inst, Stockholm, Sweden;Karolinska Univ Hosp, Stockholm, Sweden.
    Toes, Rene E. M.
    Leiden Univ, Med Ctr, Leiden, Netherlands.
    Rispens, Theo
    Univ Amsterdam, Amsterdam, Netherlands.
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Holmdahl, Rikard
    Karolinska Inst, Stockholm, Sweden;Southern Med Univ, Guangzhou, Guangdong, Peoples R China.
    Structural Basis of Cross-Reactivity of Anti-Citrullinated Protein Antibodies2019In: Arthritis & Rheumatology, ISSN 2326-5191, E-ISSN 2326-5205, Vol. 71, no 2, p. 210-221Article in journal (Refereed)
    Abstract [en]

    Objective: Anti-citrullinated protein antibodies (ACPAs) develop many years before the clinical onset of rheumatoid arthritis (RA). This study was undertaken to address the molecular basis of the specificity and cross-reactivity of ACPAs from patients with RA.

    Methods: Antibodies isolated from RA patients were expressed as monoclonal chimeric antibodies with mouse Fc. These antibodies were characterized for glycosylation using mass spectrometry, and their cross-reactivity was assessed using Biacore and Luminex immunoassays. The crystal structures of the antigen-binding fragment (Fab) of the monoclonal ACPA E4 in complex with 3 different citrullinated peptides were determined using x-ray crystallography. The prevalence of autoantibodies reactive against 3 of the citrullinated peptides that also interacted with E4 was investigated by Luminex immunoassay in 2 Swedish cohorts of RA patients.

    Results: Analysis of the crystal structures of a monoclonal ACPA from human RA serum in complex with citrullinated peptides revealed key residues of several complementarity-determining regions that recognized the citrulline as well as the neighboring peptide backbone, but with limited contact with the side chains of the peptides. The same citrullinated peptides were recognized by high titers of serum autoantibodies in 2 large cohorts of RA patients.

    Conclusion: These data show, for the first time, how ACPAs derived from human RA serum recognize citrulline. The specific citrulline recognition and backbone-mediated interactions provide a structural explanation for the promiscuous recognition of citrullinated peptides by RA-specific ACPAs.

  • 50. Gorny, Xenia
    et al.
    Mikhaylova, Marina
    Seeger, Christian
    Reddy, Pasham Parameshwar
    Reissner, Carsten
    Schott, Bjoern H.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Kreutz, Michael R.
    Seidenbecher, Constanze
    AKAP79/150 interacts with the neuronal calcium-binding protein caldendrin2012In: Journal of Neurochemistry, ISSN 0022-3042, E-ISSN 1471-4159, Vol. 122, no 4, p. 714-726Article in journal (Refereed)
    Abstract [en]

    The A kinase-anchoring protein AKAP79/150 is a postsynaptic scaffold molecule and a key regulator of signaling events. At the postsynapse it coordinates phosphorylation and dephosphorylation of receptors via anchoring kinases and phosphatases near their substrates. Interactions between AKAP79 and two Ca2+ -binding proteins caldendrin and calmodulin have been investigated here. Calmodulin is a known interaction partner of AKAP79/150 that has been shown to regulate activity of the kinase PKC in a Ca2+-dependent manner. Pull-down experiments and surface plasmon resonance biosensor analyses have been used here to demonstrate that AKAP79 can also interact with caldendrin, a neuronal calcium-binding protein implicated in regulation of Ca2+ -influx and release. We demonstrate that calmodulin and caldendrin compete for a partially overlapping binding site on AKAP79 and that their binding is differentially dependent on calcium. Therefore, this competition is regulated by calcium levels. Moreover, both proteins have different binding characteristics suggesting that the two proteins might play complementary roles. The postsynaptic enrichment, the complex binding mechanism, and the competition with calmodulin, makes caldendrin an interesting novel player in the signaling toolkit of the AKAP interactome.

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