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  • 1.
    Almlöf, Martin
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Aqvist, Johan
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Smalås, Arne O
    Brandsdal, Björn O
    Probing the effect of point mutations at protein-protein interfaces with free energy calculations.2006In: Biophys J, ISSN 0006-3495, Vol. 90, no 2, p. 433-42Article in journal (Refereed)
  • 2.
    Almlöf, Martin
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Brandsdal, Bjørn O
    Aqvist, Johan
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Binding affinity prediction with different force fields: examination of the2004In: J Comput Chem, ISSN 0192-8651, Vol. 25, no 10, p. 1242-54Article in journal (Refereed)
  • 3.
    Aqvist, Johan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Kamerlin, Shina C. L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Conserved Motifs in Different Classes of GTPases Dictate their Specific Modes of Catalysis2016In: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 6, no 3, p. 1737-1743Article in journal (Refereed)
    Abstract [en]

    The GTPase superfamily of enzymes that hydrolyze GTP have a number of conserved sequence regions (the so-called "G-motifs"), and several of the subfamilies also require catalytic activation by specific GTPase-activating proteins. In the translational GTPases involved in protein synthesis, this activating function is instead accomplished by their interaction with the ribosome. Despite these similarities, there are distinct differences regarding some of the amino acid residues making up the GTPase active sites. This raises the question of whether or not the catalytic mechanisms of different types of GTPases are identical. We report herein extensive computer simulations of both the intrinsic GTP hydrolysis reaction of Ras and the considerably faster reaction activated by the interaction with RasGAP. The results of these calculations are compared to earlier simulations of GTP hydrolysis by EF-Tu on the ribosome and show that the favored reaction pathways are strongly dependent on the composition of the active site. By computing Arrhenius plots for the temperature dependence of the calculated free energy profiles, we further show that different mechanistic pathways are associated with distinct differences in activation entropies and enthalpies. The activation parameters are in good agreement with experimental data, and we conclude that calculations of Arrhenius plots from computer simulations can be very useful for dissecting the energetics of enzyme catalysis.

  • 4.
    Aqvist, Johan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Kamerlin, Shina C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Exceptionally large entropy contributions enable the high rates of GTP hydrolysis on the ribosome2015In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 15817Article in journal (Refereed)
    Abstract [en]

    Protein synthesis on the ribosome involves hydrolysis of GTP in several key steps of the mRNA translation cycle. These steps are catalyzed by the translational GTPases of which elongation factor Tu (EF-Tu) is the fastest GTPase known. Here, we use extensive computer simulations to explore the origin of its remarkably high catalytic rate on the ribosome and show that it is made possible by a very large positive activation entropy. This entropy term (T Delta S-double dagger) amounts to more than 7 kcal/mol at 25 degrees C. It is further found to be characteristic of the reaction mechanism utilized by the translational, but not other, GTPases and it enables these enzymes to attain hydrolysis rates exceeding 500 s(-1). This entropy driven mechanism likely reflects the very high selection pressure on the speed of protein synthesis, which drives the rate of each individual GTPase towards maximal turnover rate of the whole translation cycle.

  • 5.
    Aqvist, Johan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Lind, Christoffer
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Sund, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Wallin, Goran
    Bridging the gap between ribosome structure and biochemistry by mechanistic computations2012In: Current opinion in structural biology, ISSN 0959-440X, E-ISSN 1879-033X, Vol. 22, no 6, p. 815-823Article in journal (Refereed)
    Abstract [en]

    The wealth of structural and biochemical data now available for protein synthesis on the ribosome presents major new challenges for computational biochemistry. Apart from technical difficulties in modeling ribosome systems, the complexity of the overall translation cycle with a multitude of different kinetic steps presents a formidable problem for computational efforts where we have only seen the beginning. However, a range of methodologies including molecular dynamics simulations, free energy calculations, molecular docking and quantum chemical approaches have already been put to work with promising results. In particular, the combined efforts of structural biology, biochemistry, kinetics and computational modeling can lead towards a quantitative structure-based description of translation.

  • 6.
    Azuaje, Jhonny
    et al.
    Univ Santiago de Compostela, Ctr Singular Invest Quim Biol & Mat Mol CIQUS, Santiago De Compostela 15782, Spain.;Univ Santiago de Compostela, Fac Farm, Dept Quim Organ, Santiago De Compostela 15782, Spain..
    Jespers, Willem
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Yaziji, Vicente
    Univ Santiago de Compostela, Ctr Singular Invest Quim Biol & Mat Mol CIQUS, Santiago De Compostela 15782, Spain.;Univ Santiago de Compostela, Fac Farm, Dept Quim Organ, Santiago De Compostela 15782, Spain..
    Mallo, Ana
    Univ Santiago de Compostela, Ctr Singular Invest Quim Biol & Mat Mol CIQUS, Santiago De Compostela 15782, Spain.;Univ Santiago de Compostela, Fac Farm, Dept Quim Organ, Santiago De Compostela 15782, Spain..
    Majellaro, Maria
    Univ Santiago de Compostela, Ctr Singular Invest Quim Biol & Mat Mol CIQUS, Santiago De Compostela 15782, Spain.;Univ Santiago de Compostela, Fac Farm, Dept Quim Organ, Santiago De Compostela 15782, Spain..
    Caamano, Olga
    Univ Santiago de Compostela, Fac Farm, Dept Quim Organ, Santiago De Compostela 15782, Spain..
    Loza, Maria I.
    Univ Santiago de Compostela, Ctr Singular Invest Med Mol & Enfermedades Cronic, Santiago De Compostela 15782, Spain..
    Cadavid, Maria I.
    Univ Santiago de Compostela, Ctr Singular Invest Med Mol & Enfermedades Cronic, Santiago De Compostela 15782, Spain..
    Brea, Jose
    Univ Santiago de Compostela, Ctr Singular Invest Med Mol & Enfermedades Cronic, Santiago De Compostela 15782, Spain..
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Sotelo, Eddy
    Univ Santiago de Compostela, Ctr Singular Invest Quim Biol & Mat Mol CIQUS, Santiago De Compostela 15782, Spain.;Univ Santiago de Compostela, Fac Farm, Dept Quim Organ, Santiago De Compostela 15782, Spain..
    Gutiérrez-de-Terán, Hugo
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Effect of Nitrogen Atom Substitution in A(3) Adenosine Receptor Binding: N-(4,6-Diarylpyridin-2-yl)acetamides as Potent and Selective Antagonists2017In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 60, no 17, p. 7502-7511Article in journal (Refereed)
    Abstract [en]

    We report the first family of 2-acetamidopyridines as potent and selective A, adenosine receptor (AR) antagonists. The computer -assisted design was focused on the bioisosteric replacement of the N1 atom by a CH group in a previous series of diarylpyrimidines. Some of the generated 2-acetamidopyridines elicit an antagonistic effect with excellent affinity (K-j < 10 nM) and outstanding selectivity profiles, providing an alternative and simpler chemical scaffold to the parent series of diarylpyrimidines. In addition, using molecular dynamics and free energy perturbation simulations, we elucidate the effect of the second nitrogen of the parent diarylpyrimidines, which is revealed as a stabilizer of a water network in the binding site. The discovery of 2,6-diaryl-2-acetamidopyridines represents a step forward in the search of chemically simple, potent, and selective antagonists for the hA(3)AR, and exemplifies the benefits of a joint theoretical- experimental approach to identify novel hA(3)AR antagonists through succinct and efficient synthetic methodologies.

  • 7.
    Bauer, Paul
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Barrozo, Alexandre
    Department of Chemistry, University of Southern California, SGM 418, 3620 McClintock Ave., Los Angeles, CA 90089-1062, United StatesDepartment of Chemistry, University of Southern California, SGM 418, 3620 McClintock Ave., Los Angeles, CA 90089-1062, United States.
    Purg, Miha
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Amrein, Beat Anton
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Esguerra, Mauricio
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Wilson, Philippe Barrie
    Leicester School of Pharmacy, De Montfort University, The Gateway, Leicester LE1 9BH, UK.
    Major, Dan Thomas
    Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Kamerlin, Shina C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Q6: A comprehensive toolkit for empirical valence bond and related free energy calculations2018In: SoftwareX, ISSN 2352-7110, p. 388-395Article in journal (Refereed)
    Abstract [en]

    Atomistic simulations have become one of the main approaches to study the chemistry and dynamicsof biomolecular systems in solution. Chemical modelling is a powerful way to understand biochemistry,with a number of different programs available to perform specialized calculations. We present here Q6, anew version of the Q software package, which is a generalized package for empirical valence bond, linearinteraction energy, and other free energy calculations. In addition to general technical improvements, Q6extends the reach of the EVB implementation to fast approximations of quantum effects, extended solventdescriptions and quick estimation of the contributions of individual residues to changes in the activationfree energy of reactions.

  • 8. Bjelic, Sinisa
    et al.
    Aqvist, Johan
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Catalysis and linear free energy relationships in aspartic proteases.2006In: Biochemistry, ISSN 0006-2960, Vol. 45, no 25, p. 7709-23Article in journal (Refereed)
  • 9.
    Bjelic, Sinisa
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Aqvist, Johan
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Computational prediction of structure, substrate binding mode, mechanism, and rate for a malaria protease with a novel type of active site.2004In: Biochemistry, ISSN 0006-2960, Vol. 43, no 46, p. 14521-8Article in journal (Other scientific)
  • 10.
    Boukharta, Lars
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Gutierréz de Terán, Hugo
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Computational prediction of alanine scanning and ligand binding energetics in G-protein coupled receptors2014In: PloS Computational Biology, ISSN 1553-734X, E-ISSN 1553-7358, Vol. 10, no 4, p. e1003585-Article in journal (Refereed)
    Abstract [en]

    Site-directed mutagenesis combined with binding affinity measurements is widely used to probe the nature of ligand interactions with GPCRs. Such experiments, as well as structure-activity relationships for series of ligands, are usually interpreted with computationally derived models of ligand binding modes. However, systematic approaches for accurate calculations of the corresponding binding free energies are still lacking. Here, we report a computational strategy to quantitatively predict the effects of alanine scanning and ligand modifications based on molecular dynamics free energy simulations. A smooth stepwise scheme for free energy perturbation calculations is derived and applied to a series of thirteen alanine mutations of the human neuropeptide Y1 receptor and series of eight analogous antagonists. The robustness and accuracy of the method enables univocal interpretation of existing mutagenesis and binding data. We show how these calculations can be used to validate structural models and demonstrate their ability to discriminate against suboptimal ones. Author Summary G-protein coupled receptors constitute a family of drug targets of outstanding interest, with more than 30% of the marketed drugs targeting a GPCR. The combination of site-directed mutagenesis, biochemical experiments and computationally generated 3D structural models has traditionally been used to investigate these receptors. The increasing number of GPCR crystal structures now paves the way for detailed characterization of receptor-ligand interactions and energetics using advanced computer simulations. Here, we present an accurate computational scheme to predict and interpret the effects of alanine scanning experiments, based on molecular dynamics free energy simulations. We apply the technique to antagonist binding to the neuropeptide Y receptor Y1, the structure of which is still unknown. A structural model of a Y1-antagonist complex was derived and used as starting point for computational characterization of the effects on binding of alanine substitutions at thirteen different receptor positions. Further, we used the model and computational scheme to predict the binding of a series of seven antagonist analogs. The results are in excellent agreement with available experimental data and provide validation of both the methodology and structural models of the complexes.

  • 11.
    Brandsdal, B., Österberg, F., Almlöf, M., Feierberg, I., Luzhkov, V.B.
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Åqvist, J
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Free Energy Calculations and Ligand Binding2003In: Adv. Prot. Chem., Vol. 66, p. 123-Article in journal (Refereed)
  • 12. Brandsdal, Björn O
    et al.
    Smalås, Arne O
    Åqvist, Johan
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Free energy calculations show that acidic P1 variants undergo large pKa shifts upon binding to trypsin.2006In: Proteins, ISSN 1097-0134, Vol. 64, no 3, p. 740-8Article in journal (Refereed)
  • 13.
    Brandsdal, B.O., Smalås, A.O
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Åqvist, J
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Electrostatic Effects Play a Central Role in Cold Adaptation of Trypsin2001In: FEBS Lett., Vol. 499, p. 171-Article in journal (Refereed)
  • 14.
    Brandsdal, B.O.
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Åqvist, J
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Smalås, A.O.
    Computational Analysis of Binding of P1-Variants to Trypsin2001In: Protein Sci., Vol. 10, p. 1584-Article in journal (Refereed)
  • 15. Carlsson, Jens
    et al.
    Andér, Martin
    Nervall, Martin
    Aqvist, Johan
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Continuum solvation models in the linear interaction energy method.2006In: J Phys Chem B Condens Matter Mater Surf Interfaces Biophys, ISSN 1520-6106, Vol. 110, no 24, p. 12034-41Article in journal (Refereed)
  • 16. Carlsson, Jens
    et al.
    Aqvist, Johan
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Absolute and relative entropies from computer simulation with applications to ligand binding.2005In: J Phys Chem B Condens Matter Mater Surf Interfaces Biophys, ISSN 1520-6106, Vol. 109, no 13, p. 6448-56Article in journal (Refereed)
  • 17. Carlsson, Jens
    et al.
    Åqvist, Johan
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Absolute and Relative Entropies from Computer Simulation with Applications to Ligand Binding2005In: J. Phys. Chem. B, Vol. 109, p. 6448-Article in journal (Refereed)
  • 18.
    Carlsson, Jens
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Calculations of solute and solvent entropies from molecular dynamics simulations2006In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 8, no 46, p. 5385-5395Article in journal (Refereed)
    Abstract [en]

    The translational, rotational and conformational ( vibrational) entropy contributions to ligand-receptor binding free energies are analyzed within the standard formulation of statistical thermodynamics. It is shown that the partitioning of the binding entropy into different components is to some extent arbitrary, but an appropriate method to calculate both translational and rotational entropy contributions to noncovalent association is by estimating the configurational volumes of the ligand in the bound and free states. Different approaches to calculating solute entropies using free energy perturbation calculations, configurational volumes based on root-mean-square fluctuations and covariance matrix based quasiharmonic analysis are illustrated for some simple molecular systems. Numerical examples for the different contributions demonstrate that theoretically derived results are well reproduced by the approximations. Calculation of solvent entropies, either using total potential energy averages or van't Ho. plots, are carried out for the case of ion solvation in water. Although convergence problems will persist for large and complex simulation systems, good agreement with experiment is obtained here for relative and absolute ion hydration entropies. We also outline how solvent and solute entropic contributions are taken into account in empirical binding free energy calculations using the linear interaction energy method. In particular it is shown that empirical scaling of the nonpolar intermolecular ligand interaction energy effectively takes into account size dependent contributions to the binding free energy.

  • 19.
    Carvalho, Alexandra T. P.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Szeler, Klaudia
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Vavitsas, Konstantinos
    Univ Copenhagen, Dept Plant & Environm Sci, CPSC, DK-1871 Frederiksberg C, Denmark..
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kamerlin, Lynn Shina C.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Modeling the mechanisms of biological GTP hydrolysis2015In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 582, no SI, p. 80-90Article, review/survey (Refereed)
    Abstract [en]

    Enzymes that hydrolyze GTP are currently in the spotlight, due to their molecular switch mechanism that controls many cellular processes. One of the best-known classes of these enzymes are small GTPases such as members of the Ras superfamily, which catalyze the hydrolysis of the gamma-phosphate bond in GTP. In addition, the availability of an increasing number of crystal structures of translational GTPases such as EF-Tu and EF-G have made it possible to probe the molecular details of GTP hydrolysis on the ribosome. However, despite a wealth of biochemical, structural and computational data, the way in which GTP hydrolysis is activated and regulated is still a controversial topic and well-designed simulations can play an important role in resolving and rationalizing the experimental data. In this review, we discuss the contributions of computational biology to our understanding of GTP hydrolysis on the ribosome and in small GTPases.

  • 20.
    Diwakarla, Shanti
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Nylander, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Grönbladh, Alfhild
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Reddy Vanga, Sudarsana
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Shamsudin Khan, Yasmin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Gutierrez-de-Teran, Hugo
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Ng, Leelee
    Pham, Vi
    Sävmarker, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Lundback, Thomas
    Jenmalm-Jensen, Annika
    Andersson, Hanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Engen, Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Rosenström, Ulrika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Larhed, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Yeen Chai, Siew
    Hallberg, Mathias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Binding to and Inhibition of Insulin-Regulated Aminopeptidase (IRAP) by Macrocyclic Disulfides Enhances Spine Density2016In: Molecular Pharmacology, ISSN 0026-895X, E-ISSN 1521-0111, Vol. 89, no 4, p. 413-424Article in journal (Refereed)
    Abstract [en]

    Angiotensin IV (Ang IV) and related peptide analogues, as well as non-peptide inhibitors of insulin-regulated aminopeptidase (IRAP), have previously been shown to enhance memory and cognition in animal models. Furthermore, the endogenous IRAP substrates oxytocin and vasopressin are known to facilitate learning and memory. In this study, the two recently synthesized 13-membered macrocylic competitive IRAP inhibitors HA08 and HA09, which were designed to mimic the N-terminal of oxytocin and vasopressin, were assessed and compared based on their ability to bind to the IRAP active site, and alter dendritic spine density in rat hippocampal primary cultures. The binding modes of the IRAP inhibitors HA08, HA09 and of Ang IV in either the extended or γ-turn conformation at the C-terminal to human IRAP were predicted by docking and molecular dynamics (MD) simulations. The binding free energies calculated with the linear interaction energy (LIE) method, which are in excellent agreement with experimental data and simulations, have been used to explain the differences in activities of the IRAP inhibitors, both of which are structurally very similar, but differ only with regard to one stereogenic center. In addition, we show that HA08, which is 100-fold more potent than the epimer HA09, can enhance dendritic spine number and alter morphology, a process associated with memory facilitation. Therefore, HA08, one of the most potent IRAP inhibitors known today, may serve as a suitable starting point for medicinal chemistry programs aided by MD simulations aimed at discovering more drug-like cognitive enhancers acting via augmenting synaptic plasticity.

  • 21.
    Duarte, Fernanda
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Bauer, Paul
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Barrozo, Alexandre
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Amrein, Beat Anton
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Purg, Miha
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Kamerlin, Shina Caroline Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Force Field Independent Metal Parameters Using a Nonbonded Dummy Model2014In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 118, no 16, p. 4351-4362Article in journal (Refereed)
    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.

  • 22.
    Duarte, Fernanda
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Williams, Nicholas H
    Kamerlin, Shina C Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Resolving apparent conflicts between theoretical and experimental models of phosphate monoester hydrolysis.2015In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 137, no 3, p. 1081-93Article in journal (Refereed)
    Abstract [en]

    Understanding phosphoryl and sulfuryl transfer is central to many biochemical processes. However, despite decades of experimental and computational studies, a consensus concerning the precise mechanistic details of these reactions has yet to be reached. In this work we perform a detailed comparative theoretical study of the hydrolysis of p-nitrophenyl phosphate, methyl phosphate and p-nitrophenyl sulfate, all of which have served as key model systems for understanding phosphoryl and sulfuryl transfer reactions, respectively. We demonstrate the existence of energetically similar but mechanistically distinct possibilities for phosphate monoester hydrolysis. The calculated kinetic isotope effects for p-nitrophenyl phosphate provide a means to discriminate between substrate- and solvent-assisted pathways of phosphate monoester hydrolysis, and show that the solvent-assisted pathway dominates in solution. This preferred mechanism for p-nitrophenyl phosphate hydrolysis is difficult to find computationally due to the limitations of compressing multiple bonding changes onto a 2-dimensional energy surface. This problem is compounded by the need to include implicit solvation to at least microsolvate the system and stabilize the highly charged species. In contrast, methyl phosphate hydrolysis shows a preference for a substrate-assisted mechanism. For p-nitrophenyl sulfate hydrolysis there is only one viable reaction pathway, which is similar to the solvent-assisted pathway for phosphate hydrolysis, and the substrate-assisted pathway is not accessible. Overall, our results provide a unifying mechanistic framework that is consistent with the experimentally measured kinetic isotope effects and reconciles the discrepancies between theoretical and experimental models for these biochemically ubiquitous classes of reaction.

  • 23.
    Ersmark, Karolina
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Nervall, Martin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Gutiérrez-de-Terán, Hugo
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Hamelink, Elizabeth
    Janka, Linda K.
    Clemente, Jose C.
    Dunn, Ben M.
    Gogoll, Adolf
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Samuelsson, Bertil
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Hallberg, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Macrocyclic inhibitors of the malarial aspartic proteases plasmepsin I, II, and IV2006In: Biorganic & Medicinal Chemistry, no 14, p. 2197-2208Article in journal (Refereed)
  • 24.
    Feierberg, I.
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Åqvist, J
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Correction: The Catalytic Power of Ketosteroid Isomerase Investigated by Computer Simulation2003In: Biochemistry, Vol. 42, p. 2258-Article in journal (Refereed)
  • 25.
    Feierberg, I
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Åqvist, J
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Luzhkov, V
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Computer Simulation of Primary Kinetic Isotope Effects in the Proposed Rate Limiting Step of the Glyoxalase I Reaction2000In: J. Biol. Chem., Vol. 275, p. 22657-Article in journal (Refereed)
  • 26.
    Feierberg, Isabella
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Cameron, Alexander D
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Energetics of the proposed rate-determining step of the glyoxalase I reaction1999In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 453, no 1-2, p. 90-94Article in journal (Refereed)
    Abstract [en]

    The proposed rate-limiting step of the reaction catalyzed by glyoxalase I is the proton abstraction from the C1 carbon atom of the substrate by a glutamate residue, resulting in a high-energy enolate intermediate. This proton transfer reaction was modelled using molecular dynamics and free energy perturbation simulations, with the empirical valence bond method describing the potential energy surface of the system. The calculated rate constant for the reaction is approximately 300-1500 s(-1) with Zn2+, Mg2+ or Ca2+ bound to the active site, which agrees well with observed kinetics of the enzyme. Furthermore, the results imply that the origin of the catalytic rate enhancement is mainly associated with enolate stabilization by the metal ion.

  • 27.
    Feierberg, Isabella
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Luzhkov, Victor
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Computer simulation of primary kinetic isotope effects in the proposed rate-limiting step of the glyoxalase I catalyzed reaction2000In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 275, no 30, p. 22657-22662Article in journal (Refereed)
    Abstract [en]

    The proposed rate-limiting step of the glyoxalase I catalyzed reaction is the proton abstraction from the C1 carbon of the substrate by Glu172. Here we examine primary kinetic isotope effects and the influence of quantum dynamics on this process by computer simulations. The calculations utilize the empirical valence bond method in combination with the molecular dynamics free energy perturbation technique and path integral simulations. For the enzyme-catalyzed reaction a H/D kinetic isotope effect of 5.0 ± 1.3 is predicted in reasonable agreement with the experimental result of about 3. Furthermore, the magnitude of quantum mechanical effects is found to be very similar for the enzyme reaction and the corresponding uncatalyzed process in solution, in agreement with other studies. The problems associated with attaining the required accuracy in order for the present approach to be useful as a diagnostic tool for the study of enzyme reactions are also discussed.

  • 28.
    Feierberg, Isabella
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Computational modeling of enzymatic keto-enol isomerization reactions2002In: Theoretical Chemistry accounts, ISSN 1432-881X, E-ISSN 1432-2234, Vol. 108, no 2, p. 71-84Article in journal (Refereed)
    Abstract [en]

    Catalysis of proton abstraction from nonacidic carbon atoms adjacent to a carbonyl or carboxylate group is a fundamental reaction in enzymology that has been extensively studied during the last few decades. Enzymes catalyzing these reactions, which normally involve labile enolic intermediates, need to overcome large pK a differences between the reacting groups as well as high intrinsic free-energy barriers. Here, we present an overview of results from recent computer simulation studies of keto-enol isomerization reactions catalyzed by the enzymes glyoxalase I, triosephopsphate isomerase and ketosteroid isomerase. For all three enzymes it is found that electrostatic stabilization of the transient enolate intermediates, either by charge–charge interactions or by hydrogen bonding, accounts for the main part of the activation free-energy barrier reduction. Another catalytic effect observed in all cases is the reduction of the reorganization energy by the enzyme active site. Some other factors that have been proposed to be important for these reactions are also discussed and evaluated.

  • 29.
    Feierberg, Isabella
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    The catalytic power of ketosteroid isomerase investigated by computer simulation2002In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 41, no 52, p. 15728-15735Article in journal (Refereed)
    Abstract [en]

    Ketosteroid isomerase (KSI) catalyzes the isomerization of Delta(5)-3-ketosteroids and Delta(4)-3-ketosteroids at very high rates. Here we examine the principles underlying the catalytic efficiency of KSI by computer simulations using the empirical valence bond method in combination with molecular dynamics free energy perturbation simulations. The simulations reproduce available kinetic and structural data very well and allow us to examine several features of the catalytic mechanism in detail. It is found that about 60% of the rate enhancement is due to stabilization of the negatively charged dienolate intermediate by hydrogen bonding. The critical H-bond between Tyr16 and the intermediate is found to be a normal ionic H-bond with the preferred proton location on the tyrosine residue. The remaining 40% of the catalytic effect originates from a reduction of the reorganization energy of the reaction. The possibility of an active site water molecule occupying the empty cavity adjacent to the catalytic base (Asp40) is also addressed. The existence of such a water molecule could explain how the enzyme manages to maintain a low pK(a) for the general base residue.

  • 30.
    Florian, J.
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Åqvist, J
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Warshel, A
    On the Reactivity of Phosphate Monoester Dianions in Aqueous Solution1998In: J. Am. Chem. Soc., Vol. 120, p. 11524-Article in journal (Refereed)
  • 31.
    Ge, Xueliang
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Mandava, Chandra Sekhar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Lind, Christoffer
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Complementary charge-based interaction between the ribosomal-stalk protein L7/12 and IF2 is the key to rapid subunit association2018In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 115, no 18, p. 4649-4654Article in journal (Refereed)
    Abstract [en]

    The interaction between the ribosomal-stalk protein L7/12 (L12) and initiation factor 2 (IF2) is essential for rapid subunit association, but the underlying mechanism is unknown. Here, we have characterized the L12–IF2 interaction on Escherichia coli ribosomes using site-directed mutagenesis, fast kinetics, and molecular dynamics (MD) simulations. Fifteen individual point mutations were introduced into the C-terminal domain of L12 (L12-CTD) at helices 4 and 5, which constitute the common interaction site for translational GTPases. In parallel, 15 point mutations were also introduced into IF2 between the G4 and G5 motifs, which we hypothesized as the potential L12 interaction sites. The L12 and IF2 mutants were tested in ribosomal subunit association assay in a stopped-flow instrument. Those amino acids that caused defective subunit association upon substitution were identified as the molecular determinants of L12–IF2 interaction. Further, MD simulations of IF2 docked onto the L12-CTD pinpointed the exact interacting partners—all of which were positively charged on L12 and negatively charged on IF2, connected by salt bridges. Lastly, we tested two pairs of charge-reversed mutants of L12 and IF2, which significantly restored the yield and the rate of formation of the 70S initiation complex. We conclude that complementary charge-based interaction between L12-CTD and IF2 is the key for fast subunit association. Considering the homology of the G domain, similar mechanisms may apply for L12 interactions with other translational GTPases.

  • 32.
    Graffner-Nordberg, Malin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Kolmodin, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Queener, Sherry F
    Hallberg, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Design, synthesis, and computational affinity prediction of ester soft drugs as inhibitors of dihydrofolate reductase from Pneumocystis carinii2004In: European Journal of Pharmaceutical Sciences, ISSN 0928-0987, E-ISSN 1879-0720, Vol. 22, no 1, p. 43-54Article in journal (Refereed)
    Abstract [en]

    A series of dihydrofolate reductase (DHFR) inhibitors, where the methylenamino-bridge of non-classical inhibitors was replaced with an ester function, have been prepared as potential soft drugs intended for inhalation against Pneumocystis carinii pneumonia (PCP). Several of the new ester-based inhibitors that should serve as good substrates for the ubiquitous esterases and possibly constitute safer alternatives to metabolically stable DHFR inhibitors administered orally, were found to be potent inhibitors of P. carinii DHFR (pcDHFR). Although the objectives of the present program is to achieve a favorable toxicity profile by applying the soft drug concept, a high preference for inhibition of the fungal DHFR versus the mammalian DHFR is still desirable to suppress host toxicity at the site of administration. Compounds with a slight preference for the fungal enzyme were identified. The selection of the target compounds for synthesis was partly guided by an automated docking and scoring procedure as well as molecular dynamics simulations. The modest selectivity of the synthesized inhibitors was reasonably well predicted, although a correct ranking of the relative affinities was not successful in all cases.

  • 33.
    Graffner-Nordberg, Malin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Åqvist, J
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Queener, SF
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Hallberg, A
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Kolmodin, M
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Ohlsson, S
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Andersson, P
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Design, Synthesis, Computational Prediction and Biological Evaluation of Ester Soft Drugs as Inhibitors of Dihydrofolate Reductase from Pneumocystis Carinii2001In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 44, no 15, p. 2391-2402Article in journal (Refereed)
    Abstract [en]

    A series of lipophilic soft drugs structurally related to the nonclassical dihydrofolate reductase (DHFR) inhibitors trimetrexate and piritrexim have been designed, synthesized, and evaluated in DHFR assays, with special emphasis on the inhibition of P. carinii DHFR. The best inhibitors, encompassing an ester bond in the bridge connecting the two aromatic systems, were approximately 10 times less potent than trimetrexate and piritrexim. The metabolites were designed to be poor inhibitors. Furthermore, molecular dynamics simulations of three ligands in complex with DHFR from Pneumocystis carinii and from the human enzyme were conducted in order to better understand the factors determining the selectivity. A correct ranking of the relative inhibition of DHFR was achieved utilizing the linear interaction energy method. The soft drugs are intended for local administration. One representative ester was selected for a pharmacokinetic study in rats where it was found to undergo fast metabolic degradation to the predicted inactive metabolites.

  • 34.
    Gutiérrez-de-Terán, Hugo
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Nervall, Martin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Dunn, Ben M.
    Clemente, Jose C.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Computational analysis of plasmepsin IV bound to an allophenylnorstatine inhibitor2006In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 580, no 25, p. 5910-5916Article in journal (Refereed)
    Abstract [en]

    The plasmepsin proteases from the malaria parasite Plasmodium falciparum are attracting attention as putative drug targets. A recently published crystal structure of Plasmodium malariae plasmepsin IV bound to an allophenylnorstatine inhibitor [Clemente, J.C. et al. (2006) Acta Crystallogr. D 62, 246252] provides the first structural insights regarding interactions of this family of inhibitors with plasmepsins. The compounds in this class are potent inhibitors of HIV-1 protease, but also show nM binding affinities towards plasmepsin IV. Here, we utilize automated docking, molecular dynamics and binding free energy calculations with the linear interaction energy LIE method to investigate the binding of allophenylnorstatine inhibitors to plasmepsin IV from two different species. The calculations yield excellent agreement with experimental binding data and provide new information regarding protonation states of active site residues as well as conformational properties of the inhibitor complexes.

  • 35. Gutiérrez-de-Terán, Hugo
    et al.
    Pastor, Manuel
    Centeno, Nuria B
    Aqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Sanz, Ferran
    Comparative analysis of putative agonist-binding modes in the human A1 adenosine2004In: Chembiochem, ISSN 1439-4227, Vol. 5, no 6, p. 841-9Article in journal (Refereed)
  • 36.
    Hansson, T., Marelius, J.
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Åqvist, J
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Ligand Binding Affinity Prediction by Linear Interaction Energy Methods1998In: J. Comput.-Aided Mol. Design, Vol. 12, p. 27-Article in journal (Refereed)
  • 37.
    Hansson, T., Nordlund, P.
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Åqvist, J
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Energetics of Nucleophile Activation in a Protein Tyrosine Phosphatase1997In: J. Mol. Biol., Vol. 265, p. 118-Article in journal (Refereed)
  • 38.
    Hansson, T.
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Åqvist, J
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Estimation of Binding Free Energies for HIV Proteinase Inhibitors by Molecular Dynamics Simulations1995In: Protein Eng., Vol. 8, p. 1137-Article in journal (Refereed)
  • 39.
    Henriksson, Lena M.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Unge, Torsten
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Carlsson, Jens
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Mowbray, Sherry L.
    Jones, T. Alwyn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Structures of Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate reductoisomerase provide new insights into catalysis2007In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, no 27, p. 19905-19916Article in journal (Refereed)
    Abstract [en]

    Isopentenyl diphosphate is the precursor of various isoprenoids that are essential to all living organisms. It is produced by the mevalonate pathway in humans but by an alternate route in plants, protozoa, and many bacteria. 1-Deoxy-D-xylulose-5-phosphate reductoisomerase catalyzes the second step of this non-mevalonate pathway, which involves an NADPH-dependent rearrangement and reduction of 1-deoxy-D-xylulose 5-phosphate to form 2-C-methyl-D-erythritol 4-phosphate. The use of different pathways, combined with the reported essentiality of the enzyme makes the reductoisomerase a highly promising target for drug design. Here we present several high resolution structures of the Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate reductoisomerase, representing both wild type and mutant enzyme in various complexes with Mn2+, NADPH, and the known inhibitor fosmidomycin. The asymmetric unit corresponds to the biological homodimer. Although crystal contacts stabilize an open active site in the B molecule, the A molecule displays a closed conformation, with some differences depending on the ligands bound. An inhibition study with fosmidomycin resulted in an estimated IC50 value of 80 nM. The double mutant enzyme (D151N/E222Q) has lost its ability to bind the metal and, thereby, also its activity. Our structural information complemented with molecular dynamics simulations and free energy calculations provides the framework for the design of new inhibitors and gives new insights into the reaction mechanism. The conformation of fosmidomycin bound to the metal ion is different from that reported in a previously published structure and indicates that a rearrangement of the intermediate is not required during catalysis.

  • 40. Horn, Richard
    et al.
    Roux, Benoit
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Permeation Redux: Thermodynamics and Kinetics of Ion Movement through Potassium Channels2014In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 106, no 9, p. 1859-1863Article in journal (Refereed)
    Abstract [en]

    The fundamental biophysics underlying the selective movement of ions through ion channels was launched by George Eisenman in the 1960s, using glass electrodes. This minireview examines the insights from these early studies and the explosive progress made since then.

  • 41. Isaksen, Geir Villy
    et al.
    Andberg, Tor Arne Heim
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Brandsdal, Bjorn Olav
    Qgui: A high-throughput interface for automated setup and analysis of free energy calculations and empirical valence bond simulations in biological systems2015In: Journal of Molecular Graphics and Modelling, ISSN 1093-3263, E-ISSN 1873-4243, Vol. 60, p. 15-23Article in journal (Refereed)
    Abstract [en]

    Structural information and activity data has increased rapidly for many protein targets during the last decades. In this paper, we present a high-throughput interface (Qgui) for automated free energy and empirical valence bond (EVB) calculations that use molecular dynamics (MD) simulations for conformational sampling. Applications to ligand binding using both the linear interaction energy (LIE) method and the free energy perturbation (FEP) technique are given using the estrogen receptor (ER alpha) as a model system. Examples of free energy profiles obtained using the EVB method for the rate-limiting step of the enzymatic reaction catalyzed by trypsin are also shown. In addition, we present calculation of high-precision Arrhenius plots to obtain the thermodynamic activation enthalpy and entropy with Qgui from running a large number of EVB simulations.

  • 42.
    Isaksen, Geir Villy
    et al.
    Univ Tromso, Fac Sci & Technol, Dept Chem, Ctr Theoret & Computat Chem, N-9037 Tromso, Norway..
    Hopmann, Kathrin Helen
    Univ Tromso, Fac Sci & Technol, Dept Chem, Ctr Theoret & Computat Chem, N-9037 Tromso, Norway..
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Brandsdal, Björn Olav
    Univ Tromso, Fac Sci & Technol, Dept Chem, Ctr Theoret & Computat Chem, N-9037 Tromso, Norway..
    Computer Simulations Reveal Substrate Specificity of Glycosidic Bond Cleavage in Native and Mutant Human Purine Nucleoside Phosphorylase2016In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 55, no 14, p. 2153-2162Article in journal (Refereed)
    Abstract [en]

    Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine ribonucleosides and 2'-deoxyribonucleosides, yielding the purine base and (2'deoxy)ribose 1-phosphate as products. While this enzyme has been extensively studied, several questions with respect to the catalytic mechanism have remained largely unanswered. The role of the phosphate and key amino acid residues in the catalytic reaction as well as the purine ring protonation state is elucidated using density functional theory calculations and extensive empirical valence bond (EVB) simulations. Free energy surfaces for adenosine, inosine, and guanosine are fitted to ab initio data and yield quantitative agreement with experimental data when the surfaces are used to model the corresponding enzymatic reactions. The cognate substrates 6-aminopurines (inosine and guanosine) interact with PNP through extensive hydrogen bonding, but the substrate specificity is found to be a direct result of the electrostatic preorganization energy along the reaction coordinate. Asn243 has previously been identified as a key residue providing substrate specificity. Mutation of Asn243 to Asp has dramatic effects on the substrate specificity, making 6-amino- and 6-oxopurines equally good as substrates. The principal effect of this particular mutation is the change in the electrostatic preorganization energy between the native enzyme and the Asn243Asp mutant, clearly favoring adenosine over inosine and guanosine. Thus, the EVB simulations show that this particular mutation affects the electrostatic preorganization of the active site, which in turn can explain the substrate specificity.

  • 43. Isaksen, Geir Villy
    et al.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Brandsdal, Bjorn Olav
    Protein Surface Softness Is the Origin of Enzyme Cold-Adaptation of Trypsin2014In: PloS Computational Biology, ISSN 1553-734X, E-ISSN 1553-7358, Vol. 10, no 8, p. e1003813-Article in journal (Refereed)
    Abstract [en]

    Life has effectively colonized most of our planet and extremophilic organisms require specialized enzymes to survive under harsh conditions. Cold-loving organisms (psychrophiles) express heat-labile enzymes that possess a high specific activity and catalytic efficiency at low temperatures. A remarkable universal characteristic of cold-active enzymes is that they show a reduction both in activation enthalpy and entropy, compared to mesophilic orthologs, which makes their reaction rates less sensitive to falling temperature. Despite significant efforts since the early 1970s, the important question of the origin of this effect still largely remains unanswered. Here we use cold-and warm-active trypsins as model systems to investigate the temperature dependence of the reaction rates with extensive molecular dynamics free energy simulations. The calculations quantitatively reproduce the catalytic rates of the two enzymes and further yield high-precision Arrhenius plots, which show the characteristic trends in activation enthalpy and entropy. Detailed structural analysis indicates that the relationship between these parameters and the 3D structure is reflected by significantly different internal protein energy changes during the reaction. The origin of this effect is not localized to the active site, but is found in the outer regions of the protein, where the cold-active enzyme has a higher degree of softness. Several structural mechanisms for softening the protein surface are identified, together with key mutations responsible for this effect. Our simulations further show that single point-mutations can significantly affect the thermodynamic activation parameters, indicating how these can be optimized by evolution.

  • 44.
    Isaksen, Geir Villy
    et al.
    Univ Tromso, Dept Chem, Ctr Theoret & Computat Chem, NO-9037 Tromso, Norway..
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Brandsdal, Björn Olav
    Univ Tromso, Dept Chem, Ctr Theoret & Computat Chem, NO-9037 Tromso, Norway..
    Enzyme surface rigidity tunes the temperature dependence of catalytic rates2016In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 113, no 28, p. 7822-7827Article in journal (Refereed)
    Abstract [en]

    The structural origin of enzyme adaptation to low temperature, allowing efficient catalysis of chemical reactions even near the freezing point of water, remains a fundamental puzzle in biocatalysis. A remarkable universal fingerprint shared by all cold-active enzymes is a reduction of the activation enthalpy accompanied by a more negative entropy, which alleviates the exponential decrease in chemical reaction rates caused by lowering of the temperature. Herein, we explore the role of protein surface mobility in determining this enthalpy-entropy balance. The effects of modifying surface rigidity in cold-and warm-active trypsins are demonstrated here by calculation of high-precision Arrhenius plots and thermodynamic activation parameters for the peptide hydrolysis reaction, using extensive computer simulations. The protein surface flexibility is systematically varied by applying positional restraints, causing the remarkable effect of turning the cold-active trypsin into a variant with mesophilic characteristics without changing the amino acid sequence. Furthermore, we show that just restraining a key surface loop causes the same effect as a point mutation in that loop between the cold-and warm-active trypsin. Importantly, changes in the activation enthalpy-entropy balance of up to 10 kcal/mol are almost perfectly balanced at room temperature, whereas they yield significantly higher rates at low temperatures for the cold-adapted enzyme.

  • 45.
    Isaksen, Geir Villy
    et al.
    Univ Tromso, Dept Chem, Ctr Theoret & Computat Chem, NO-9037 Tromso, Norway..
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Brandsdal, Björn Olav
    Univ Tromso, Dept Chem, Ctr Theoret & Computat Chem, NO-9037 Tromso, Norway..
    Thermodynamics of the Purine Nucleoside Phosphorylase Reaction Revealed by Computer Simulations2017In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 56, no 1, p. 306-312Article in journal (Refereed)
    Abstract [en]

    Enzymes are able to catalyze chemical reactions by reducing the activation free energy, yielding significant increases in the reaction rates. This can thermodynamically be accomplished by either reducing the activation enthalpy or increasing the activation entropy. The effect of remote mutations on the thermodynamic activation parameters of human purine nucleoside phosphorylase is examined using extensive molecular dynamics and free energy simulations. More than 2700 independent reaction free energy profiles for six different temperatures have been calculated to obtain high-precision computational Arrhenius plots. On the basis of these, the activation enthalpies and entropies were computed from linear regression of the plots with Delta G(double dagger) as a function of 1/T, and the obtained thermodynamic activation parameters are in very good agreement with those from experiments. The Arrhenius plots immediately show that the 6-oxopurines (INO and GUO) have identical slopes, whereas the 6-aminopurine (ADO) has a significantly different slope, indicating that the substrate specificity is related to the difference in thermodynamic activation parameters. Furthermore, the calculations show that the human PNP specificity for 6-oxopurines over 6-aminopurines originates from significant differences in electrostatic preorganization. The effect of the remote double mutation, K22E and H104R (E:R), has also been examined, as it alters human PNP toward the bovine PNP. These residues are situated on the protein surface, 28-35 angstrom from the active site, and the mutation alters the enthalpy entropy balance with little effect on the catalytic rates. It is thus quite remarkable that the empirical valence bond method can reproduce the enthalpies and entropies induced by these long-range mutations.

  • 46.
    Isaksson, Rebecka
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preparative Medicinal Chemistry.
    Lindman, Jens
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preparative Medicinal Chemistry.
    Wannberg, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Sallander, Jessica
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Backlund, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Baraldi, Dhaniel
    Department of Pharmacology, Monash University.
    Widdop, Robert
    Department of Pharmacology, Monash University.
    Hallberg, Mathias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Gutierrez de Teran, Hugo
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Gising, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preparative Medicinal Chemistry.
    Larhed, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preparative Medicinal Chemistry.
    A Series of Analogues to the AT2R Prototype Antagonist C38 Allow Fine Tuning of the Previously Reported Antagonist Binding Mode2019In: ChemistryOpen, ISSN 2191-1363, Vol. 8, no 1, p. 114-125Article in journal (Refereed)
    Abstract [en]

    We here report on our continued studies of ligands binding tothe promising drug target angiotensin II type 2 receptor (AT2R). Two series of compounds were synthesized and investigated. The first series explored the effects of adding small substituents to the phenyl ring of the known selective nonpeptide AT2R antagonist C38, generating small but significant shifts in AT2R affinity. One compound in the first series was equipotent to C38 and showed similar kinetic solubility, and stability in both human and mouse liver microsomes. The second series was comprised of new bicyclic derivatives, amongst which one ligand exhibited a five-fold improved affinity to AT2R ascompared to C38. The majority of the compounds in the second series, including the most potent ligand, were inferior to C38 with regard to stability in both human and mouse microsomes. In contrast to our previously reported findings, ligands with shorter carbamate alkyl chains only demonstrated slightly improved stability in microsomes. Based on data presented herein, a more adequate, tentative model of the binding modes of ligand analogues to the prototype AT2R antagonist C38 is proposed, as deduced from docking redefined by molecular dynamic simulations.

  • 47.
    Jespers, Willem
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Esguerra, Mauricio
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Gutiérrez-de-Terán, Hugo
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    QligFEP: an automated workflow for small molecule free energy calculations in Q2019In: Journal of Cheminformatics, ISSN 1758-2946, E-ISSN 1758-2946, Vol. 11, article id 26Article in journal (Refereed)
    Abstract [en]

    The process of ligand binding to a biological target can be represented as the equilibrium between the relevant solvated and bound states of the ligand. This which is the basis of structure-based, rigorous methods such as the estimation of relative binding affinities by free energy perturbation (FEP). Despite the growing capacity of computing power and the development of more accurate force fields, a high throughput application of FEP is currently hampered due to the need, in the current schemes, of an expert user definition of the alchemical transformations between molecules in the series explored. Here, we present QligFEP, a solution to this problem using an automated workflow for FEP calculations based on a dual topology approach. In this scheme, the starting poses of each of the two ligands, for which the relative affinity is to be calculated, are explicitly present in the MD simulations associated with the (dual topology) FEP transformation, making the perturbation pathway between the two ligands univocal. We show that this generalized method can be applied to accurately estimate solvation free energies for amino acid sidechain mimics, as well as the binding affinity shifts due to the chemical changes typical of lead optimization processes. This is illustrated in a number of protein systems extracted from other FEP studies in the literature: inhibitors of CDK2 kinase and a series of A(2A) adenosine G protein-coupled receptor antagonists, where the results obtained with QligFEP are in excellent agreement with experimental data. In addition, our protocol allows for scaffold hopping perturbations to identify the binding affinities between different core scaffolds, which we illustrate with a series of Chk1 kinase inhibitors. QligFEP is implemented in the open-source MD package Q, and works with the most common family of force fields: OPLS, CHARMM and AMBER.

  • 48.
    Jespers, Willem
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Oliveira, Ana
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Prieto-Diaz, Ruben
    Univ Santiago Compostela, Fac Farm, Dept Quim Organ, Ctr Singular Invest Quim Biol & Mat Mol CIQUS, Santiago De Compostela 15782, Spain..
    Majellaro, Maria
    Univ Santiago Compostela, Fac Farm, Dept Quim Organ, Ctr Singular Invest Quim Biol & Mat Mol CIQUS, Santiago De Compostela 15782, Spain..
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Sotelo, Eddy
    Univ Santiago Compostela, Fac Farm, Dept Quim Organ, Ctr Singular Invest Quim Biol & Mat Mol CIQUS, Santiago De Compostela 15782, Spain..
    Gutiérrez-de-Terán, Hugo
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Structure-Based Design of Potent and Selective Ligands at the Four Adenosine Receptors2017In: Molecules, ISSN 1420-3049, E-ISSN 1420-3049, Vol. 22, no 11, article id 1945Article in journal (Refereed)
    Abstract [en]

    The four receptors that signal for adenosine, A(1), A(2A), A(2B) and A(3) ARs, belong to the superfamily of G protein-coupled receptors (GPCRs). They mediate a number of (patho)physiological functions and have attracted the interest of the biopharmaceutical sector for decades as potential drug targets. The many crystal structures of the A(2A), and lately the A(1) ARs, allow for the use of advanced computational, structure-based ligand design methodologies. Over the last decade, we have assessed the efficient synthesis of novel ligands specifically addressed to each of the four ARs. We herein review and update the results of this program with particular focus on molecular dynamics (MD) and free energy perturbation (FEP) protocols. The first in silico mutagenesis on the A(1)AR here reported allows understanding the specificity and high affinity of the xanthine-antagonist 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX). On the A(2A)AR, we demonstrate how FEP simulations can distinguish the conformational selectivity of a recent series of partial agonists. These novel results are complemented with the revision of the first series of enantiospecific antagonists on the A(2B)AR, and the use of FEP as a tool for bioisosteric design on the A(3)AR.

  • 49.
    Kazemi, Masoud
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Himo, Fahmi
    Stockholm Univ, Arrhenius Lab, Dept Organ Chem, SE-10691 Stockholm, Sweden..
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Enzyme catalysis by entropy without Circe effect2016In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 113, no 9, p. 2406-2411Article in journal (Refereed)
    Abstract [en]

    Entropic effects have often been invoked to explain the extraordinary catalytic power of enzymes. In particular, the hypothesis that enzymes can use part of the substrate-binding free energy to reduce the entropic penalty associated with the subsequent chemical transformation has been very influential. The enzymatic reaction of cytidine deaminase appears to be a distinct example. Here, substrate binding is associated with a significant entropy loss that closely matches the activation entropy penalty for the uncatalyzed reaction inwater, whereas the activation entropy for the rate-limiting catalytic step in the enzyme is close to zero. Herein, we report extensive computer simulations of the cytidine deaminase reaction and its temperature dependence. The energetics of the catalytic reaction is first evaluated by density functional theory calculations. These results are then used to parametrize an empirical valence bond description of the reaction, which allows efficient sampling by molecular dynamics simulations and computation of Arrhenius plots. The thermodynamic activation parameters calculated by this approach are in excellent agreement with experimental data and indeed show an activation entropy close to zero for the rate-limiting transition state. However, the origin of this effect is a change of reaction mechanism compared the uncatalyzed reaction. The enzyme operates by hydroxide ion attack, which is intrinsically associated with a favorable activation entropy. Hence, this has little to do with utilization of binding free energy to pay the entropic penalty but rather reflects how a preorganized active site can stabilize a reaction path that is not operational in solution.

  • 50.
    Kazemi, Masoud
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Himo, Fahmi
    Stockholm Univ, Arrhenius Lab, Dept Organ Chem, SE-10691 Stockholm, Sweden..
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Peptide Release on the Ribosome Involves Substrate-Assisted Base Catalysis2016In: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 6, no 12, p. 8432-8439Article in journal (Refereed)
    Abstract [en]

    Termination of protein synthesis on the ribosome involves hydrolysis of the ester bond between the P-site tRNA and the nascent peptide chain. This reaction occurs in the peptidyl transferase center and is triggered by the class I release factors RF1 and RF2 in prokaryotes. Peptidyl-tRNA hydrolysis is pH-dependent, and experimental results suggest that an ionizable group with pK(a) > 9 is involved in the reaction. The nature of this group is, however, unknown. To resolve this problem, we conducted density functional theory calculations using a large cluster model of the peptidyl transferase center. Our calculations reveal that peptidyl-tRNA hydrolysis occurs via a base-catalyzed mechanism with a predicted activation energy of 15.8 kcal mol(-1), which is in good agreement with experimental data. In this mechanism, the P-site A76 2'-OH group is deprotonated and acts as the general base by activating the nucleophilic water molecule. The energy cost of deprotonating the 2'-hydroxyl group at pH 7.5 is estimated to be about 8 kcal mo1(-1), on the basis of its experimental plc in aqueous solution, and this step is predicted to be the source of the observed pH dependence. The proposed mechanism is consistent not only with experimentally derived activation energies but also with the observed kinetic solvent isotope effect.

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