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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.
    Calixto, Ana Rita
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Univ Porto, Dept Quim & Bioquim, UCIBIO REQUIMTE, Fac Ciencias, Rua Campo Alegre S-N, P-4169007 Porto, Portugal.
    Ramos, Maria Joao
    Univ Porto, Dept Quim & Bioquim, UCIBIO REQUIMTE, Fac Ciencias, Rua Campo Alegre S-N, P-4169007 Porto, Portugal.
    Fernandes, Pedro Alexandrino
    Univ Porto, Dept Quim & Bioquim, UCIBIO REQUIMTE, Fac Ciencias, Rua Campo Alegre S-N, P-4169007 Porto, Portugal.
    Conformational diversity induces nanosecond-timescale chemical disorder in the HIV-1 protease reaction pathway2019In: Chemical Science, ISSN 2041-6520, E-ISSN 2041-6539, Vol. 10, no 30, p. 7212-7221Article in journal (Refereed)
    Abstract [en]

    The role of conformational diversity in enzyme catalysis has been a matter of analysis in recent studies. Pre-organization of the active site has been pointed out as the major source for enzymes' catalytic power. Following this line of thought, it is becoming clear that specific, instantaneous, non-rare enzyme conformations that make the active site perfectly pre-organized for the reaction lead to the lowest activation barriers that mostly contribute to the macroscopically observed reaction rate. The present work is focused on exploring the relationship between structure and catalysis in HIV-1 protease (PR) with an adiabatic mapping method, starting from different initial structures, collected from a classical MD simulation. The first, rate-limiting step of the HIV-1 PR catalytic mechanism was studied with the ONIOM QM/MM methodology (B3LYP/6-31G(d):ff99SB), with activation and reaction energies calculated at the M06-2X/6-311++G(2d,2p):ff99SB level of theory, in 19 different enzyme:substrate conformations. The results showed that the instantaneous enzyme conformations have two independent consequences on the enzyme's chemistry: they influence the barrier height, something also observed in the past in other enzymes, and they also influence the specific reaction pathway, which is something unusual and unexpected, challenging the "one enzyme-one substrate-one reaction mechanism" paradigm. Two different reaction mechanisms, with similar reactant probabilities and barrier heights, lead to the same gem-diol intermediate. Subtle nanosecond-timescale rearrangements in the active site hydrogen bonding network were shown to determine which reaction the enzyme follows. We named this phenomenon chemical disorder. The results make us realize the unexpected mechanistic consequences of conformational diversity in enzymatic reactivity.

  • 25.
    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)
  • 26.
    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.

  • 27.
    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)