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  • 201.
    Eklund, Sandra
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Interpreting a Giant: Studies of Structure and Function of Tripeptidyl-peptidase II2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Tripeptidyl-peptidase II (TPP II) is a subtilisin-like serine peptidase that forms a gigantic homooligomeric complex, and is involved in the degradation of peptides in the cytosol. In addition, TPP II has been implicated in specific cellular processes, such as apoptosis control and adipogenesis, but if this is dependent on its endo- or exopeptidase activity remains to be determined. This work is devoted to the structure and function of TPP II, and to finding connections between the two.

    Evolutionarily conserved regions of TPP II have been identified, and sequence signatures have been constructed as an aid in identification of TPP II homologues. The conserved regions highlight amino acid residues of potential importance to structure, function or both. In addition, the first TPP II homologue in a prokaryote has been documented, which was likely the result of a horizontal gene transfer.

    Substrate binding for the exopeptidase activity of TPP II has been studied through mutagenesis of Glu-331, which revealed a molecular ruler mechanism that positions substrates for cleavage at the third peptide bond from the N-terminus. Thus, the well-known tripeptidyl-releasing property of TPP II could be explained. The exopeptidase activity was also probed by pH dependence studies, which revealed that a substrate with a smaller residue in the P1 position could bind non-productively to the active site. Furthermore, a difference in the pH dependence of KM between TPP II from Drosophila and homologues from mammals indicated a difference in the configuration of the binding pockets between these species.

    The endopeptidase activity of TPP II has also been investigated, and was found to differ from the exopeptidase activity. The endopeptidase activity appeared to be promiscuous and the preference for basic amino acid residues in the P1 position reported earlier could not be substantiated.

    In conclusion, many structural and mechanistic features have been observed in this work. This might be of value to future drug discovery efforts towards TPP II, and in elucidating the physiological role of this gigantic enzyme.

    List of papers
    1. Development, evaluation and application of tripeptidyl-peptidase II sequence signatures
    Open this publication in new window or tab >>Development, evaluation and application of tripeptidyl-peptidase II sequence signatures
    2009 (English)In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 484, no 1, p. 39-45Article in journal (Refereed) Published
    Abstract [en]

    Tripeptidyl-peptidase II (TPP II) is a cytosolic peptidase that has been implicated in fat formation and cancer, apparently independent of the enzymatic activity. In search for alternative functional regions, conserved motifs were identified and eleven signatures were constructed. Seven of the signatures covered previously investigated residues, whereas the functional importance of the other motifs is unknown. This provides directions for future investigations of alternative activities of TPP II. The obtained signatures provide an efficient bioinformatic tool for the identification of TPP II homologues. Hence, a TPP II sequence homologue from fission yeast, Schizosaccharomyces pombe, was identified and demonstrated to encode the TPP II-like protein previously reported as multicorn. Furthermore, an homologous protein was found in the prokaryote Blastopirellula marina, albeit the TPP II function was apparently not conserved. This gene is probably the result of a rare gene transfer from eukaryote to prokaryote.

    Keywords
    Serine protease, subtilase, sequence motif, cytosolic protein degradation, tripeptidyl-peptidase II
    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-119529 (URN)10.1016/j.abb.2009.01.007 (DOI)000264927700006 ()19467630 (PubMedID)
    Available from: 2010-02-26 Created: 2010-02-26 Last updated: 2017-12-12
    2. Investigation of a role for Glu-331 and Glu-305 in substrate binding of tripeptidyl-peptidase II
    Open this publication in new window or tab >>Investigation of a role for Glu-331 and Glu-305 in substrate binding of tripeptidyl-peptidase II
    2008 (English)In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1784, no 12, p. 1899-1907Article in journal (Refereed) Published
    Abstract [en]

    The aim of this study was to investigate the mechanism by which tripeptidyl-peptidase II (TPP II) can specifically release tripeptides from the free N-terminus of an oligopeptide. The subtilisin-like N-terminal part of TPP II was modelled using subtilisin as template. Two glutamate residues (Glu-305 and Glu-331) appeared to be positioned so as to interact with the positively charged N-terminus of the substrate. In order to test this potential interaction, both residues were replaced by glutamine and lysine. The catalytic efficiency was reduced 400-fold for the E331Q variant and 20000-fold for the E331K variant, compared with the wild-type (wt). A substantial part of this reduction was due to decreased substrate affinity, since the K(M) for both mutants was at least two orders of magnitude greater than for the wt. This decrease was linked specifically to interaction with the free N-terminal amino group, based on inhibition studies. Glu-305 appears to be essential for enzymatic activity, but the extremely low activity of the E305Q variant prevented an investigation of the involvement of Glu-305 in substrate binding. The present work is, to our knowledge, the first report to investigate a mechanism for a tripeptidyl-peptidase activity through site-directed mutagenesis.

    Keywords
    Tripeptidyl-peptidase II, Exopeptidase, Substrate specificity, Intracellular protein degradation, Oligomerization, Molecular ruler
    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-100055 (URN)10.1016/j.bbapap.2008.08.017 (DOI)000261673300002 ()18822395 (PubMedID)
    Available from: 2009-03-24 Created: 2009-03-24 Last updated: 2017-12-13Bibliographically approved
    3. Inter-species variation in the pH dependence of tripeptidyl-peptidase II
    Open this publication in new window or tab >>Inter-species variation in the pH dependence of tripeptidyl-peptidase II
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Tripeptidyl-peptidase II (TPP II) is a large enzyme complex (>4 MDa) participating in the general protein turn-over in the cell downstream of the proteasome. In addition, there have been reports of involvement of TPP II in different physiological situations. To facilitate further investigations of the physiological role of TPP II and its enzymatic properties, a characterization at protein level is necessary. Therefore, an expression system for murine TPP II using Escherichia coli has been developed. The pH-optimum for cleavage of two different chromogenic substrates, Ala-Ala-Phe-pNA and Ala-Ala-Ala-pNA, was investigated for mTPP II, and compared with human TPP II and TPP II from Drosophila melanogaster. It was shown that the mouse enzyme had similar pH dependence as the human enzyme, while dTPP II had a slightly lower optimum. Surprisingly, the investigation also demonstrated that TPP II from all sources showed a different pH-profile for hydrolysis of AAA-pNA compared to AAF-pNA. To investigate this observation further, steady-state kinetic parameters were determined at various pH. Since both the KM and Vmax are lower for cleavage of AAA-pNA, a potential explanation could be that the substrate AAA-pNA is non-productively bound to the active site of the enzyme.

    Keywords
    tripeptidyl-peptidase II, TPP II, AAF-pNA, AAA-pNA, steady-state kinetics, pH-dependence
    National Category
    Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Biochemistry and Molecular Biology
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-134621 (URN)
    Available from: 2010-11-30 Created: 2010-11-30 Last updated: 2011-06-28
    4. Characterization of the endopeptidase activity of tripeptidyl-peptidase II
    Open this publication in new window or tab >>Characterization of the endopeptidase activity of tripeptidyl-peptidase II
    Show others...
    2012 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 424, no 3, p. 503-507Article in journal (Refereed) Published
    Abstract [en]

    Tripeptidyl-peptidase II (TPP II) is a giant cytosolic peptidase with a proposed role in cellular protein degradation and protection against apoptosis. Beside its well-characterised exopeptidase activity, TPP II also has an endopeptidase activity. Little is known about this activity, and since it could be important for the physiological role of TPP II, we have investigated it in more detail. Two peptides, Nef(69-87) and LL37, were incubated with wild-type murine TPP II and variants thereof as well as TPP II from human and Drosophila melanogaster. Two intrinsically disordered proteins were also included in the study. We conclude that the endopeptidase activity is more promiscuous than previously reported. It is also clear that TPP II can attack longer disordered peptides up to 75 amino acid residues. Using a novel FRET substrate, the catalytic efficiency of the endopeptidase activity could be determined to be 5 orders of magnitude lower than for the exopeptidase activity.

    Keywords
    TPP II, endopeptidase, alternate activity
    National Category
    Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Biochemistry and Molecular Biology
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-134620 (URN)10.1016/j.bbrc.2012.06.144 (DOI)000307618800024 ()
    Available from: 2010-11-30 Created: 2010-11-30 Last updated: 2017-12-12Bibliographically approved
  • 202.
    Eklund, Sandra
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Dogan, Jakob
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kalbacher, Hubert
    Interfaculty institute of Biochemistry, University of Tübingen.
    Tomkinson, Birgitta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Characterization of the endopeptidase activity of tripeptidyl-peptidase II2012In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 424, no 3, p. 503-507Article in journal (Refereed)
    Abstract [en]

    Tripeptidyl-peptidase II (TPP II) is a giant cytosolic peptidase with a proposed role in cellular protein degradation and protection against apoptosis. Beside its well-characterised exopeptidase activity, TPP II also has an endopeptidase activity. Little is known about this activity, and since it could be important for the physiological role of TPP II, we have investigated it in more detail. Two peptides, Nef(69-87) and LL37, were incubated with wild-type murine TPP II and variants thereof as well as TPP II from human and Drosophila melanogaster. Two intrinsically disordered proteins were also included in the study. We conclude that the endopeptidase activity is more promiscuous than previously reported. It is also clear that TPP II can attack longer disordered peptides up to 75 amino acid residues. Using a novel FRET substrate, the catalytic efficiency of the endopeptidase activity could be determined to be 5 orders of magnitude lower than for the exopeptidase activity.

  • 203.
    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.

  • 204.
    Eklund, Sandra
    et al.
    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 Organismal Biology, Molecular Evolution.
    Hamnevik, Emil
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Tomkinson, Birgitta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Inter-species variation in the pH dependence of tripeptidyl-peptidase IIManuscript (preprint) (Other academic)
    Abstract [en]

    Tripeptidyl-peptidase II (TPP II) is a large enzyme complex (>4 MDa) participating in the general protein turn-over in the cell downstream of the proteasome. In addition, there have been reports of involvement of TPP II in different physiological situations. To facilitate further investigations of the physiological role of TPP II and its enzymatic properties, a characterization at protein level is necessary. Therefore, an expression system for murine TPP II using Escherichia coli has been developed. The pH-optimum for cleavage of two different chromogenic substrates, Ala-Ala-Phe-pNA and Ala-Ala-Ala-pNA, was investigated for mTPP II, and compared with human TPP II and TPP II from Drosophila melanogaster. It was shown that the mouse enzyme had similar pH dependence as the human enzyme, while dTPP II had a slightly lower optimum. Surprisingly, the investigation also demonstrated that TPP II from all sources showed a different pH-profile for hydrolysis of AAA-pNA compared to AAF-pNA. To investigate this observation further, steady-state kinetic parameters were determined at various pH. Since both the KM and Vmax are lower for cleavage of AAA-pNA, a potential explanation could be that the substrate AAA-pNA is non-productively bound to the active site of the enzyme.

  • 205.
    Eklöf, Anders M.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Lowcoordinated Silicon and Hypercoordinated Carbon: Structure and Stability of Silicon Analogs of Alkenes and Carbon Analogs of Silicates2008Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Quantum chemical studies on lowcoordinated group 14-16 compounds have been performed. This thesis focuses particularly on silenes influenced by reverse Siδ-=Cδ+ bond polarization. Hypercoordinated carbon compounds are also studied.

    The geometries from calculations with several common computationally inexpensive methods have been tested against high level CCSD/cc-pVTZ geometries for a series of substituted silenes. Hybrid HF/DFT methods performed best among the inexpensive methods tested for silenes.

    Heavy alkenes strongly influenced by reverse polarization are found to have less exothermic dimerization energies for both head-to-head and head-to-tail dimerizations, and to have higher activation energies for water addition than naturally polarized heavy alkenes.

    We also investigated solvated lithium, magnesium and potassium silenolates and found that lithium and magnesium ions coordinate preferably to O, giving their SiC bond some double bond character.

    Reverse polarized 2-siloxy-, 2-thiosiloxy-, and 2-(N-sila-N-methyl)-silenes could according to calculations be formed thermolytically from the corresponding tetrasilanes as transient species. It was, however, found that silenes highly influenced by π-conjugative reverse polarization have low barriers for the back-reaction, and thus these silenes are more difficult to form as stable species than naturally polarized silenes.

    It is also found that conjugated 1-siladienes, formed by electrocyclic ring-opening of 1-silacyclobut-2-enes, which are highly influenced by π-conjugative reverse polarization, have higher barriers for electrocyclization back to starting material than naturally polarized 1-siladienes.

    It is found that CHe54+, CHe64+, CNe54+, and CNe64+ are the closest carbon analogs of SiH5-, SiH62-, SiF5- and SiF62-, respectively. However, due to their exothermic dissociation reaction, these very high-lying local minima will be impossible to reach experimentally.

    List of papers
    1. An Assessment of the Performance of Inexpensive Quantum Chemical Methods for the Calculation of Substituted Silenes and Stannenes
    Open this publication in new window or tab >>An Assessment of the Performance of Inexpensive Quantum Chemical Methods for the Calculation of Substituted Silenes and Stannenes
    Manuscript (Other academic)
    Identifiers
    urn:nbn:se:uu:diva-97584 (URN)
    Available from: 2008-10-03 Created: 2008-10-03 Last updated: 2010-01-13Bibliographically approved
    2. On the Role of the π-Contribution to Reverse Polarization for Structure and Stability of Heavy group 14-16 Alkene, Imine, and Carbonyl analogs
    Open this publication in new window or tab >>On the Role of the π-Contribution to Reverse Polarization for Structure and Stability of Heavy group 14-16 Alkene, Imine, and Carbonyl analogs
    In: Journal of the American Chemical SocietyArticle in journal (Refereed) Submitted
    Identifiers
    urn:nbn:se:uu:diva-97585 (URN)
    Available from: 2008-10-03 Created: 2008-10-03Bibliographically approved
    3. Effects of Substituents and Counterions on the Structures of Silenolates: A Computational Investigation
    Open this publication in new window or tab >>Effects of Substituents and Counterions on the Structures of Silenolates: A Computational Investigation
    2009 (English)In: Tetrahedron, ISSN 0040-4020, E-ISSN 1464-5416, Vol. 65, no 28, p. 5521-5526Article in journal (Refereed) Published
    Abstract [en]

    The structures and charge distributions of substituted silenolates   [H2SiC(=O)X](-) (X-H, SiH3, Me, t-Bu, OMe, NMe2; group A),  [Y2SiC(=O)H](-) (Y=H, F, Me, Ph, SiH3, SiMe3; group B), and [Y2SiC(=O)X](-) (Y=Me, X=t-Bu, and Y=SiMe3; X=t-Bu, OMe, NMe2; group C)   were examined through density functional theory calculations. The effects of the solvated counterion (K+, Li+, or MgCl+) and coordination site (O or Si) on the properties of group C silenolates were also Studied. The variation in the degree of pi-conjugative reverse SiC bond   polarization, Sigma Phi(RP)(pi) calculated by natural resonance theory,   was determined. The Sigma Phi(RP)(pi) correlated with r(SiC) for both   group A and B silenolates, and the correlation between Sigma   Phi(RP)(pi) and the Sum of valence angles at Si, Sigma alpha(Si), was   good for group A but poor for group B due to strong influence of the   inductive effect. The SiC charge difference correlated well with Sigma   Phi(RP)(pi) for group A, but not for group B, again an effect of   inductive substituent effects. The group C silenolates were Coordinated   to Li(THF)(3)(+), MgCl(THF)(4)(+), and K(THF)(5)(+) either via the O or   Si atom. The coordination energies show that coordination to the hard O   is preferred for Li+ and MgCl+, but the K+ ion coordinated   simultaneously to Si and O. Coordination of the solvated metal ion to O  resulted in shorter SiC bond length, an increased Sigma alpha(Si)   value, and lower Delta q(SiC) when compared to the naked silenolate.  Choice Of counterion and substituent provides a means to extensively vary the properties of silenolates such as their reactivity.

    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-97586 (URN)10.1016/j.tet.2009.02.088 (DOI)000268128000010 ()
    Available from: 2008-10-03 Created: 2008-10-03 Last updated: 2017-12-14Bibliographically approved
    4. Relation between the π-Contribution to Reversed Si═C Bond Polarization and the Reaction Profile for the Thermolytic Formation of Silenes
    Open this publication in new window or tab >>Relation between the π-Contribution to Reversed Si═C Bond Polarization and the Reaction Profile for the Thermolytic Formation of Silenes
    2008 (English)In: Organometallics, ISSN 0276-7333, E-ISSN 1520-6041, Vol. 27, no 20, p. 5203-5211Article in journal (Refereed) Published
    Abstract [en]

    A quantum chemical investigation of the reaction profiles for the thermal formation of silenes Z2Si═C(XSiH3)Y from silanes Z2(H3Si)Si−C(═X)Y (X = O, S, NMe; Y = NMe2, OMe, SMe, Me; Z = SiH3, Me) has been performed. Focus was put on the influence of the π-conjugative contribution to reversed Si═C bond polarization (Siδ−═Cδ+) as determined by natural resonance theory (NRT) at the B3LYP density functional theory level. Good linear correlations between the weights of π-conjugated reverse polarized resonance structures (ΣΦRP(π)) in the electronic structure and the Si═C bond lengths were found for the two classes of silenes with Z = SiH3 and Me (r2 = 0.957 and 0.955, respectively). Silenes that are strongly influenced by the π-conjugative reverse polarization have low barriers for back-reaction to the silanes, making these silenes more difficult to isolate when formed through a [1,3]-silyl shift than those that are naturally polarized. Modest exponential dependencies of the activation barriers for the reverse reactions on ΣΦRP(π) are found (r2 = 0.685 for Z = SiH3 and r2 = 0.699 for Z = Me). Species with the silyl groups replaced by trimethylsilyl groups, e.g., the Brook-type silene (Me3Si)2Si═C(OSiMe3)t-Bu, have lower contributions of ΣΦRP(π) by 3−23% than the corresponding model silenes, a result of steric bulk. The weight ΣΦRP(π) to the electronic structure of (Me3Si)2Si═C(OSiMe3)t-Bu was calculated to be 7.4%. With Z = Me, the silenes are in general not equally influenced by ΣΦRP(π) as with Z = SiH3, their energies relative to the silanes are higher, and they have higher activation barriers for both forward and backward reactions.

    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:uu:diva-97587 (URN)10.1021/om800477j (DOI)
    Available from: 2008-10-03 Created: 2008-10-03 Last updated: 2017-12-14Bibliographically approved
    5. A Computational Investigation of the Retrocyclization Reaction of Silacyclo-but-2-enes to 1-silabuta-1,3-dienes: Focus on the effect of the substituents
    Open this publication in new window or tab >>A Computational Investigation of the Retrocyclization Reaction of Silacyclo-but-2-enes to 1-silabuta-1,3-dienes: Focus on the effect of the substituents
    2007 (English)In: Journal of Molecular Structure: THEOCHEM, ISSN 0166-1280, Vol. 811, no 1-3, p. 153-160Article in journal (Refereed) Published
    Abstract [en]

    Substituent effects on the reaction profile of the thermal retrocyclization reaction of silacyclobut-2-enes to 1-silabuta-1,3-dienes were studied using B3LYP hybrid density functional theory as well as CCSD and CCSD(T) ab initio calculations. Several different substituents (–CF3, –SiH3, –CN, –OCH3, –OH, and –NH2) were used to investigate their effects on the relative energies of the transition states of the retrocyclization reaction as well as of the 1-silabutadiene products. It was found that π-donor groups at the 4-position greatly reduce the energy barriers, and also stabilize the 1-silabutadienes relative to the silacyclobut-2-enes. Silyl substituents at the silicon atom will facilitate the reaction when compared to alkyl substituents. The results thus indicate that the ring-opening reaction of 4,4-disubstituted 1,1-disilylsilacyclobut-2-enes with π-donor substituents are particularly suitable entries for formation of 1-silabutadienes of low relative energy.

    Keywords
    Silicon, Retrocyclization, Density functional theory, Ab initio, Silene, Silacyclobut-2-ene, 1-Silabutadiene
    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-97588 (URN)10.1016/j.theochem.2007.02.024 (DOI)000247053500018 ()
    Available from: 2008-10-03 Created: 2008-10-03 Last updated: 2017-12-14Bibliographically approved
  • 206.
    Eklöf, Anders M.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Guliashvili, Tamaz
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Ottosson, Henrik
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Relation between the π-Contribution to Reversed Si═C Bond Polarization and the Reaction Profile for the Thermolytic Formation of Silenes2008In: Organometallics, ISSN 0276-7333, E-ISSN 1520-6041, Vol. 27, no 20, p. 5203-5211Article in journal (Refereed)
    Abstract [en]

    A quantum chemical investigation of the reaction profiles for the thermal formation of silenes Z2Si═C(XSiH3)Y from silanes Z2(H3Si)Si−C(═X)Y (X = O, S, NMe; Y = NMe2, OMe, SMe, Me; Z = SiH3, Me) has been performed. Focus was put on the influence of the π-conjugative contribution to reversed Si═C bond polarization (Siδ−═Cδ+) as determined by natural resonance theory (NRT) at the B3LYP density functional theory level. Good linear correlations between the weights of π-conjugated reverse polarized resonance structures (ΣΦRP(π)) in the electronic structure and the Si═C bond lengths were found for the two classes of silenes with Z = SiH3 and Me (r2 = 0.957 and 0.955, respectively). Silenes that are strongly influenced by the π-conjugative reverse polarization have low barriers for back-reaction to the silanes, making these silenes more difficult to isolate when formed through a [1,3]-silyl shift than those that are naturally polarized. Modest exponential dependencies of the activation barriers for the reverse reactions on ΣΦRP(π) are found (r2 = 0.685 for Z = SiH3 and r2 = 0.699 for Z = Me). Species with the silyl groups replaced by trimethylsilyl groups, e.g., the Brook-type silene (Me3Si)2Si═C(OSiMe3)t-Bu, have lower contributions of ΣΦRP(π) by 3−23% than the corresponding model silenes, a result of steric bulk. The weight ΣΦRP(π) to the electronic structure of (Me3Si)2Si═C(OSiMe3)t-Bu was calculated to be 7.4%. With Z = Me, the silenes are in general not equally influenced by ΣΦRP(π) as with Z = SiH3, their energies relative to the silanes are higher, and they have higher activation barriers for both forward and backward reactions.

  • 207.
    Eklöf, Anders M
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Gullashvili, Tarnaz
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Ottosson, Henrik
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Relation between the pi-Contribution to Reversed Si=C Bond Polarization and the Reaction Profile for the Thermolytic Formation of Silenes2008In: Organometallics, ISSN 0276-7333, E-ISSN 1520-6041, Vol. 27, no 20, p. 5203-5211Article in journal (Refereed)
    Abstract [en]

    A quantum chemical investigation of the reaction profiles for the thermal formation of silenes Z(2)Si=C(XSiH3)Y from silanes Z(2)(H3Si)Si-C(=X)Y (X = O, S, NMe; Y = NMe2, OMe, SMe, Me; Z = SiH3, Me) has been performed. Focus was put on the influence of the pi-conjugative contribution to reversed Si=C bond polarization (Si delta-=C delta+) as determined by natural resonance theory (NRT) at the B3LYP density functional theory level. Good linear correlations between the weights of pi-conjugated reverse polarized resonance structures (Sigma Phi(RP)(pi)) in the electronic structure and the Si=C bond lengths were found for the two classes of silenes with Z = SiH3 and Me (r(2) = 0.957 and 0.955, respectively). Silenes that are strongly influenced by the pi-conjugative reverse polarization have low barriers for back-reaction to the silanes, making these silenes more difficult to isolate when formed through a [1,3]-silyl shift than those that are naturally polarized. Modest exponential dependencies of the activation barriers for the reverse reactions on Sigma Phi(RP)(pi) are found (r(2) = 0.685 for Z = SiH3 and r(2) = 0.699 for Z = Me). Species with the silyl groups replaced by trimethylsilyl groups, e.g., the Brook-type silene (Me3Si)(2)Si=C(OSi-Me-3)t-Bu, have lower contributions of Sigma Phi(RP)(pi) by 3-23% than the corresponding model silenes, a result of steric bulk. The weight Sigma Phi(RP)(pi) to the electronic structure of (Me3Si)(2)Si=C(OSiMe3)t-Bu was calculated to be 7.4%. With Z = Me, the silenes are in general not equally influenced by Sigma Phi(RP)(pi) as with Z = SiH3, their energies relative to the silanes are higher, and they have higher activation barriers for both forward and backward reactions.

  • 208.
    Eklöf, Anders M
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Ottosson, Henrik
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Effects of Substituents and Counterions on the Structures of Silenolates: A Computational Investigation2009In: Tetrahedron, ISSN 0040-4020, E-ISSN 1464-5416, Vol. 65, no 28, p. 5521-5526Article in journal (Refereed)
    Abstract [en]

    The structures and charge distributions of substituted silenolates   [H2SiC(=O)X](-) (X-H, SiH3, Me, t-Bu, OMe, NMe2; group A),  [Y2SiC(=O)H](-) (Y=H, F, Me, Ph, SiH3, SiMe3; group B), and [Y2SiC(=O)X](-) (Y=Me, X=t-Bu, and Y=SiMe3; X=t-Bu, OMe, NMe2; group C)   were examined through density functional theory calculations. The effects of the solvated counterion (K+, Li+, or MgCl+) and coordination site (O or Si) on the properties of group C silenolates were also Studied. The variation in the degree of pi-conjugative reverse SiC bond   polarization, Sigma Phi(RP)(pi) calculated by natural resonance theory,   was determined. The Sigma Phi(RP)(pi) correlated with r(SiC) for both   group A and B silenolates, and the correlation between Sigma   Phi(RP)(pi) and the Sum of valence angles at Si, Sigma alpha(Si), was   good for group A but poor for group B due to strong influence of the   inductive effect. The SiC charge difference correlated well with Sigma   Phi(RP)(pi) for group A, but not for group B, again an effect of   inductive substituent effects. The group C silenolates were Coordinated   to Li(THF)(3)(+), MgCl(THF)(4)(+), and K(THF)(5)(+) either via the O or   Si atom. The coordination energies show that coordination to the hard O   is preferred for Li+ and MgCl+, but the K+ ion coordinated   simultaneously to Si and O. Coordination of the solvated metal ion to O  resulted in shorter SiC bond length, an increased Sigma alpha(Si)   value, and lower Delta q(SiC) when compared to the naked silenolate.  Choice Of counterion and substituent provides a means to extensively vary the properties of silenolates such as their reactivity.

  • 209.
    Eklöf, Anders M.
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry.
    Ottosson, Henrik
    On the Role of the π-Contribution to Reverse Polarization for Structure and Stability of Heavy group 14-16 Alkene, Imine, and Carbonyl analogsIn: Journal of the American Chemical SocietyArticle in journal (Refereed)
  • 210.
    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.

  • 211. Ekström, Ulf
    et al.
    Ottosson, Henrik
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Norrman, Patrick
    Characterization of the Chemisorption of Methylsilane on a Au(1,1,1) Surface from the Silicon K- and L-Edge Spectra: A Theoretical Study Using the Four-Component Static Exchange Approximation2007In: The Journal of Physical Chemistry C, ISSN 1932-7447, E-ISSN 1932-7455, Vol. 111, no 37, p. 13846-13850Article in journal (Refereed)
    Abstract [en]

    X-ray photoelectron spectroscopy (XPS) and near-edge X-ray absorption fine structure spectra (NEXAFS) of methylsilane, isolated and chemisorbed to a Au(1,1,1) surface, are determined in the fully relativistic four-component static exchange approximationboth the K- and the L-edge of silicon are addressed in this investigation. In the fully chemisorbed structure, three H(Si) atoms have been cleaved off when Si binds in the hollow site of Au forming three Si−Au bonds of normal length. As due to the tri-coordinated chemisorption, the onsets of the K- and L-edge NEXAFS absorption bands occur some 2.0 and 2.5 eV lower in energy, respectively. The spin−orbit splittings in the silicon 2p-shell are not significantly changed due to adsorption. A partly chemisorbed methylsilane with only one H(Si) bond cleaved was also studied, and it is shown that the polarization dependence in the surface spectra contains details that can be used experimentally to identify the surface coordination of silicon. The red-shifts in the XPS silicon 1s (2p) spectra upon surface binding are 0.95 (0.65) and 1.15 (0.83) eV for the mono- and tricoordinated system, respectively.

  • 212.
    Elfström, Lisa
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Catalysis of potato epoxide hydrolase, StEH12005In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 390, p. 633-640Article in journal (Refereed)
    Abstract [en]

    The kinetic mechanism of epoxide hydrolase (EC 3.3.2.3) from potato, StEH1 (Solanum tuberosum epoxide hydrolase 1), was studied by presteady-state and steady-state kinetics as well as by pH dependence of activity. The specific activities towards the different enantiomers of TSO (trans-stilbene oxide) as substrate were 43 and 3 mmol·min-1·mg-1 with the R,R- or S,S-isomers respectively. The enzyme was, however, enantioselective in favour of the S,S enantiomer due to a lower Km value. The pH dependences of kcat with R,R or S,S-TSO were also distinct and supposedly reflecting the pH dependences of the individual kinetic rates during substrate conversion. The rate-limiting step for TSO and cis- and trans-epoxystearate was shown by rapid kinetic measurements to be the hydrolysis of the alkylenzyme intermediate. Functional characterization of point mutants verified residues Asp105, Tyr154, Tyr235 and His300 as crucial for catalytic activity. All mutants displayed drastically decreased enzymatic activities during steady state. Presteady-state measurements revealed the base-deficient H300N (His300Asn) mutant to possess greatly reduced efficiencies in catalysis of both chemical steps (alkylation and hydrolysis).

  • 213.
    Elfström, Lisa
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    The Saccharomyces cerevisiae ORF YNR064c protein has characteristics of an 'orphaned' epoxide hydrolase2005In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1748, p. 213-221Article in journal (Refereed)
    Abstract [en]

    The open reading frame YNR064c in Saccharomyces cerevisiae encodes a protein tentatively assigned as similar to a bacterialdehalogenase. In this study we conclude that the YNR064c protein displays characteristics of an epoxide hydrolase belonging to the a/hhydrolasefold family of enzymes. Endogenous expression of the protein in S. cerevisiae was confirmed and a His-tagged variant of theprotein was heterologously expressed in both Escherichia coli and Pichia pastoris for isolation and characterization. The YNR064c proteindisplayed low but reproducible epoxide hydrolase activity with racemic phenanthrene 9,10-oxide and trans- or cis-stilbene oxide.Phylogenetic analysis of related gene products found in various microorganisms suggested that the YNR064c protein is a member of a newsubclass of a/h-hydrolase fold enzymes.

  • 214.
    Elinder, Malin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Towards a New Generation of Anti-HIV Drugs: Interaction Kinetic Analysis of Enzyme Inhibitors Using SPR-biosensors2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    As of today, there are 25 drugs approved for the treatment of HIV and AIDS. Nevertheless, HIV continues to infect and kill millions of people every year. Despite intensive research efforts, both a vaccine and a cure remain elusive and the long term efficacy of existing drugs is limited by the development of resistant HIV strains. New drugs and preventive strategies that are effective against resistant virus are therefore still needed. In this thesis an enzymological approach, primarily using SPR-based interaction kinetic analysis, has been used for identification and characterization of compounds of potential use in next generation anti-HIV drugs.

    By screening of a targeted non-nucleoside reverse transcriptase inhibitor (NNRTI) library, one novel and highly potent NNRTI was identified. The inhibitor was selected with respect to resilience to drug resistance and for high affinity and slow dissociation – a kinetic profile assumed to be suitable for inhibitors used in topical microbicides. In order to confirm the hypothesis that such a kinetic profile would result in an effective preventive agent with long-lasting effect, the correlation between antiviral effect and kinetic profile was investigated for a panel of NNRTIs. The kinetic profiles revealed that NNRTI efficacy is dependent on slow dissociation from the target, although the induced fit interaction mechanism prevented quantification of the rate constants.

    To avoid cross-resistance, the next generation anti-HIV drugs should be based on chemical entities that do not resemble drugs in clinical use, either in structure or mode-of-action. Fragment-based drug discovery was used for identification of structurally new inhibitors of HIV-enzymes. One fragment that was effective also on variants of HIV RT with resistance mutations was identified. The study revealed the possibility of identifying structurally novel NNRTIs as well as fragments interacting with other sites of the protein.

    The two compounds identified in this thesis represent potential starting points for a new generation of NNRTIs. The applied methodologies also show how interaction kinetic analysis can be used as an effective and versatile tool throughout the lead discovery process, especially when integrated with functional enzymological assays.

    List of papers
    1. Screening for NNRTIs with Slow Dissociation and High Affinity for a Panel of HIV-1 RT Variants
    Open this publication in new window or tab >>Screening for NNRTIs with Slow Dissociation and High Affinity for a Panel of HIV-1 RT Variants
    Show others...
    2009 (English)In: Journal of Biomolecular Screening, ISSN 1087-0571, E-ISSN 1552-454X, Vol. 14, no 4, p. 395-403Article in journal (Refereed) Published
    Abstract [en]

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

    Place, publisher, year, edition, pages
    SAGE, 2009
    Keywords
    HIV-1 Reverse transcriptase, screening, surface plasmon resonance (SPR)-biosensor, NNRTIs, microbicides
    National Category
    Chemical Sciences
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-109362 (URN)10.1177/1087057109333977 (DOI)000265948100008 ()19403922 (PubMedID)
    Available from: 2009-10-22 Created: 2009-10-14 Last updated: 2018-06-26Bibliographically approved
    2. Inhibition of HIV-1 by non-nucleoside reverse transcriptase inhibitors via an induced fit mechanism: Importance of slow dissociation and relaxation rates for antiviral efficacy
    Open this publication in new window or tab >>Inhibition of HIV-1 by non-nucleoside reverse transcriptase inhibitors via an induced fit mechanism: Importance of slow dissociation and relaxation rates for antiviral efficacy
    Show others...
    2010 (English)In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 80, no 8, p. 1133-1140Article in journal (Refereed) Published
    Abstract [en]

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

    Keywords
    HIV reverse transcriptase, NNRTI, MIV, kinetics, biosensor, SPR
    National Category
    Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
    Identifiers
    urn:nbn:se:uu:diva-129428 (URN)10.1016/j.bcp.2010.06.035 (DOI)000281936800003 ()20599774 (PubMedID)
    Available from: 2010-08-15 Created: 2010-08-15 Last updated: 2018-06-26Bibliographically approved
    3. Experimental Validation of a Fragment Library for Lead Discovery Using SPR Biosensor Technology
    Open this publication in new window or tab >>Experimental Validation of a Fragment Library for Lead Discovery Using SPR Biosensor Technology
    Show others...
    2011 (English)In: Journal of Biomolecular Screening, ISSN 1087-0571, E-ISSN 1552-454X, Vol. 16, no 1, p. 15-25Article in journal (Refereed) Published
    Abstract [en]

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

    Keywords
    enzyme, fragment library, fragment screening, interaction analysis, SPR biosensor
    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-140679 (URN)10.1177/1087057110389038 (DOI)000286063300002 ()21149860 (PubMedID)
    Available from: 2011-01-07 Created: 2011-01-07 Last updated: 2018-06-26Bibliographically approved
    4. Identification of a Novel Scaffold for Allosteric Inhibition of Wild Type and Drug Resistant HIV-1 Reverse Transcriptase by Fragment Library Screening
    Open this publication in new window or tab >>Identification of a Novel Scaffold for Allosteric Inhibition of Wild Type and Drug Resistant HIV-1 Reverse Transcriptase by Fragment Library Screening
    Show others...
    2011 (English)In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 54, no 3, p. 699-708Article in journal (Refereed) Published
    Abstract [en]

    A novel scaffold inhibiting wild type and drug resistant variants of human immunodeficiency virus type 1 reverse transcriptase (HIV-1RT) has been identified in a library consisting of 1040 fragments. The fragments were significantly different from already known non-nucleoside reverse transcriptase inhibitors (NNRTIs), as indicated by a Tversky similarity analysis. A screening strategy involving SPR biosensor-based interaction analysis and enzyme inhibition was used. Primary biosensor-based screening, using short concentration series, was followed by analysis of nevirapine competition and enzyme inhibition, thus identifying inhibitory fragments binding to the non-nucleoside reverse transcriptase inhibitor (NNRTI) binding site. Ten hits were discovered, and their affinities and resistance profiles were evaluated with wild type and three drug resistant enzyme variants (K103N, Y181C, and L100I). One fragment exhibited submillimolar K(D) and IC(50) values against all four tested enzyme variants. A substructure comparison between the fragment and 826 structurally diverse published NNRTIs confirmed that the scaffold was novel. The fragment is a bromoindanone with a ligand efficiency of 0.42 kcal/mol(-1).

    National Category
    Other Basic Medicine
    Identifiers
    urn:nbn:se:uu:diva-140680 (URN)10.1021/jm1010513 (DOI)000286798100002 ()21207961 (PubMedID)
    Available from: 2011-01-07 Created: 2011-01-07 Last updated: 2018-06-26Bibliographically approved
  • 215.
    Elinder, Malin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Danielson, Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Unge, Torsten
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    VanHam, Guido
    Öberg, Bo
    MIV-170: A novel NNRTI exhibiting tight binding to HIV-1 reverse transcriptase (RT)2008Conference paper (Refereed)
    Abstract [en]

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

  • 216.
    Elinder, Malin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Geitmann, Matthis
    Gossas, Thomas
    Källblad, Per
    Winquist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Nordström, Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Hämäläinen, Markkuu D.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Experimental Validation of a Fragment Library for Lead Discovery Using SPR Biosensor Technology2011In: Journal of Biomolecular Screening, ISSN 1087-0571, E-ISSN 1552-454X, Vol. 16, no 1, p. 15-25Article in journal (Refereed)
    Abstract [en]

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

  • 217.
    Elinder, Malin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Nordström, Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Geitmann, Matthis
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Hämäläinen, Markku
    Vrang, Lotta
    Öberg, Bo
    Danielson, U Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Screening for NNRTIs with Slow Dissociation and High Affinity for a Panel of HIV-1 RT Variants2009In: Journal of Biomolecular Screening, ISSN 1087-0571, E-ISSN 1552-454X, Vol. 14, no 4, p. 395-403Article in journal (Refereed)
    Abstract [en]

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

  • 218.
    Elinder, Malin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Selhorst, Philippe
    Vanham, Guido
    Öberg, Bo
    Vrang, Lotta
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Inhibition of HIV-1 by non-nucleoside reverse transcriptase inhibitors via an induced fit mechanism: Importance of slow dissociation and relaxation rates for antiviral efficacy2010In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 80, no 8, p. 1133-1140Article in journal (Refereed)
    Abstract [en]

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

  • 219.
    Ellfolk, Maria
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Norlin, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Isolation and properties of the CYP2D25 promoter: Transcriptional regulation by vitamin D3 metabolites2006In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 345, no 2, p. 568-572Article in journal (Refereed)
    Abstract [en]

    Previous studies have suggested that hepatic production of 25-hydroxyvitamin D3 may be suppressed by 1α,25-dihydroxyvitamin D3. However, the molecular details of these observations have not been clarified. In the current study, the 5´-flanking DNA sequence of CYP2D25, a porcine microsomal vitamin D 25-hydroxylase, was isolated and analyzed. The CYP2D25 promoter contains a putative vitamin D response element (VDRE). The promoter activity was markedly suppressed by 1α,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 in presence of vitamin D receptor (VDR). The data suggest that VDR-mediated inhibition of 25-hydroxylase(s) by vitamin D3 metabolites at the transcriptional level may play an important role in the regulation of 25-hydroxyvitamin D3 production in liver and other tissues.

  • 220. El-Nahas, Ahmed
    et al.
    Sandström, Niclas
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry.
    Ottosson, Henrik
    Will Heavy-Core Staffanes Function as Hole transporting Mo-lecular Wires? A Computational InvestigationManuscript (Other academic)
  • 221.
    Emrén, Lars O.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Kurtovic, Sanela
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Runarsdottir, Arna
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Larsson, Anna-Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Functionally diverging molecular quasi-species evolve by crossing two enzymes2006In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 103, no 29, p. 10866-10870Article in journal (Refereed)
    Abstract [en]

    Molecular evolution is frequently portrayed by structural relationships, but delineation of separate functional species is more elusive. We have generated enzyme variants by stochastic recombinations of DNA encoding two homologous detoxication enzymes, human glutathione transferases M1-1 and M2-2, and explored their catalytic versatilities. Sampled mutants were screened for activities with eight alternative substrates, and the activity fingerprints were subjected to principal component analysis. This phenotype characterization clearly identified at least three distributions of substrate selectivity, where one was orthogonal to those of the parent-like distributions. This approach to evolutionary data mining serves to identify emerging molecular quasi-species and indicates potential trajectories available for further protein evolution.

  • 222. Enander, Karin
    et al.
    Aili, D.
    Baltzer, Lars
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry II.
    Lundström, I.
    Liedberg, B.
    Alpha helix-inducing dimerization of synthetic plypeptide scaffolds on gold2005In: Langmuir, no 21, p. 2480-2487Article in journal (Refereed)
  • 223. Enander, Karin
    et al.
    Aili, D.
    Baltzer, Lars
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Chemistry. Department of Biochemistry and Organic Chemistry, Organic Chemistry II.
    Lundström, I.
    Liedberg, B.
    Alpha helix-inducing dimerization of synthetic polypeptide scaffolds on gold2005In: Langmuir, no 21, p. 2480-2487Article in journal (Refereed)
  • 224.
    Enander, Karin
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Chemistry. Department of Biochemistry and Organic Chemistry, Organic Chemistry II.
    Dolphin, G. T.
    Baltzer, Lars
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Chemistry. Department of Biochemistry and Organic Chemistry, Organic Chemistry II.
    Designed functionalized helix-loop-helix motifs that bind human Carbonic Anhydrase II- a new class of synthetic receptor molecules2004In: J. Am. Chem. Soc., no 126, p. 4464-4465Article in journal (Refereed)
  • 225.
    Enander, Karin
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Chemistry. Department of Biochemistry and Organic Chemistry, Organic Chemistry II.
    Dolphin, G.T.
    Liedberg, B.
    Lundström, I.
    Baltzer, Lars
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Chemistry. Department of Biochemistry and Organic Chemistry, Organic Chemistry II.
    A versatile polypeptide platform for intergrated recognition and reporting - affinity arrays for protein - ligand interaction analysis2004In: Chem. Eur. J., no 10, p. 2375-2385Article in journal (Refereed)
  • 226. Enander, Karin
    et al.
    Dolphin, Gunnar T.
    Baltzer, Lars
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry II.
    Designed, functionalized helix-loop-helix motifs that bind human Carbonic Anhydrase II - a new class of synthetic receptor molecules2004In: J. Am. Chem. Soc., no 126, p. 4464-4465Article in journal (Refereed)
  • 227. Enander, Karin
    et al.
    Dolphin, Gunnar T.
    Liedberg, Bo
    Lundström, Ingemar
    Baltzer, Lars
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry II.
    A versatile polypeptide platform for integrated recognition and reporting - affinity arrays for protein-ligand interaction2004In: Chem. Eur. J., no 10, p. 2375-2385Article in journal (Refereed)
  • 228. Eng, W.
    et al.
    Atack, J. R.
    Bergström, M.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Section of Nuclear Medicine and PET.
    Sanabria, S.
    Appel, Lieuwe
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Section of Nuclear Medicine and PET.
    Dawson, G. R.
    Sciberras, D.
    Hargreaves, R. J.
    Långström, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Burns, H. D.
    Occupancy of human brain GABA(A) receptors by the novel alpha 5 subtype-selective benzodiazepine site inverse agonist alpha 5IA as measured using [C-11]flumazenil PET imaging2010In: Neuropharmacology, ISSN 0028-3908, E-ISSN 1873-7064, Vol. 59, no 7-8, p. 635-639Article in journal (Refereed)
    Abstract [en]

    GABA(A) receptor alpha 5-selective inverse agonists enhance cognitive performance in pre-clinical species. However, a key aspect of the clinical development of such compounds is the demonstration that in man such compounds are devoid of the anxiogenic-like activity associated with non-selective inverse agonists such as FG 7142. The triazolophthalazine alpha 5IA (3-(5-methylisoxazol-3-yl)-6-[(1-methyl-1,2,3-triazol-4-yl) methyloxy]-1,2,4-triazolo[3,4-a]phthalazine) is an alpha 5-selective inverse agonist which enhances cognitive performance in rodents and encouragingly in human Phase I Safety and Tolerability studies it was devoid of the anxiogenic-like activity associated with FG 7142. However, in order to appropriately interpret this latter observation, it was considered important to demonstrate that the absence of anxiogenic-like activity occurs at significant levels of receptor occupancy. Consequently, the occupancy of human brain GABAA receptors was measured using [C-11]flumazenil positron emission tomography in three healthy normal young male volunteers following a single oral dose of 2 mg alpha 5IA. One hour after dosing, mean occupancy levels were 53% and this fell to 16% by 8 h post-dose, with the plasma alpha 5IA concentration corresponding to 50% occupancy being 10 ng/mL. These data clearly show that an alpha 5-selective inverse agonist is not associated with anxiogenic-like side effects at doses that give 50% occupancy.

  • 229.
    Engberg, Anna E.
    et al.
    Linnéuniversitetet.
    Rosengren-Holmberg, Jenny P.
    Linnéuniversitetet.
    Chen, Hui
    University of Pennsylvania.
    Nilsson, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology.
    Lambris, John D.
    University of Pennsylvania.
    Nicholls, Ian A.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Nilsson Ekdahl, Kristina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology.
    Blood protein-polymer adsorption: Implications for understanding complement-mediated hemoincompatibility2011In: Journal of Biomedical Materials Research - Part A, ISSN 1549-3296, Vol. 97A, no 1, p. 74-84Article in journal (Refereed)
    Abstract [en]

    The aim of this study was to create polymeric materials with known properties to study the preconditions for complement activation. Initially, 22 polymers were screened for complement activating capacity. Based on these results, six polymers (P1-P6) were characterized regarding physico-chemical parameters, for example, composition, surface area, pore size, and protein adsorption from human EDTA-plasma. P2, P4, and reference particles of polystyrene and polyvinyl chloride, were hydrophobic, bound low levels of protein and were poor complement activators. Their accessible surface was limited to protein adsorption in that they had pore diameters smaller than most plasma proteins. P1 and P3 were negatively charged and adsorbed IgG and C1q. A 10-fold difference in complement activation was attributed to the fact that P3 but not P1 bound high amounts of C1-inhibitor. The hydrophobic P5 and P6 were low complement activators. They selectively bound apolipoproteins AI and AIV (and vitronectin), which probably limited the binding of complement activators to the surface. We demonstrate the usefulness of the modus operandi to use a high-throughput procedure to synthesize a great number of novel substances, assay their physico-chemical properties with the aim to study the relationship between the initial protein coat on a surface and subsequent biological events. Data obtained from the six polymers characterized here, suggest that a complement-resistant surface should be hydrophobic, uncharged, and have a small available surface, accomplished by nanostructured topography. Additional attenuation of complement can be achieved by selective enrichment of inert proteins and inhibitors.

  • 230.
    Engdahl, Carin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry.