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Isaksson, R., Lindman, J., Wannberg, J., Sallander, J., Backlund, M., Baraldi, D., . . . Larhed, M. (2019). A Series of Analogues to the AT2R Prototype Antagonist C38 Allow Fine Tuning of the Previously Reported Antagonist Binding Mode. ChemistryOpen, 8(1), 114-125
Open this publication in new window or tab >>A Series of Analogues to the AT2R Prototype Antagonist C38 Allow Fine Tuning of the Previously Reported Antagonist Binding Mode
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2019 (English)In: ChemistryOpen, ISSN 2191-1363, Vol. 8, no 1, p. 114-125Article in journal (Refereed) Published
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.

National Category
Organic Chemistry Medicinal Chemistry
Identifiers
urn:nbn:se:uu:diva-377050 (URN)10.1002/open.201800282 (DOI)000457433000017 ()30697513 (PubMedID)
Funder
Swedish National Infrastructure for Computing (SNIC)Swedish Research Council
Available from: 2019-02-13 Created: 2019-02-13 Last updated: 2019-04-04Bibliographically approved
Lind, C., Esguerra, M., Jespers, W., Satpati, P., Gutiérrez-de-Terán, H. & Åqvist, J. (2019). Free energy calculations of RNA interactions. Methods, 162-163, 85-95
Open this publication in new window or tab >>Free energy calculations of RNA interactions
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2019 (English)In: Methods, ISSN 1046-2023, E-ISSN 1095-9130, Vol. 162-163, p. 85-95Article in journal (Refereed) Published
Abstract [en]

This review discusses the use of molecular dynamics free energy calculations for characterizing RNA interactions, with particular emphasis on molecular recognition events involved in mRNA translation on the ribosome. The general methodology for efficient free energy calculations is outlined and our specific implementation for binding free energy changes due to base mutations in mRNA and tRNA is described, We show that there are a number of key problems related to the accuracy of protein synthesis that can be addressed with this type of computational approach and several such examples are discussed in detail. These include the decoding of mRNA during peptide chain elongation, initiation and termination of translation, as well as the energetic effects of base tautomerization and tRNA modifications. It is shown that free energy calculations can be made sufficiently reliable to allow quantitative conclusions to be drawn regarding the energetics of cognate versus non-cognate interactions and its structural origins.

Place, publisher, year, edition, pages
Elsevier, 2019
Keywords
Free energy calculation, Molecular dynamics, RNA interactions, Ribosome, mRNA translation
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-390696 (URN)10.1016/j.ymeth.2019.02.014 (DOI)000475837300011 ()30794905 (PubMedID)
Funder
Swedish Research CouncilKnut and Alice Wallenberg FoundationSwedish National Infrastructure for Computing (SNIC)
Available from: 2019-08-13 Created: 2019-08-13 Last updated: 2019-08-13Bibliographically approved
Ge, X., Oliveira, A., Hjort, K., Bergfors, T., Gutiérrez-de-Terán, H., Andersson, D. I., . . . Åqvist, J. (2019). Inhibition of translation termination by small molecules targeting ribosomal release factors. Scientific Reports, 9, Article ID 15424.
Open this publication in new window or tab >>Inhibition of translation termination by small molecules targeting ribosomal release factors
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2019 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 15424Article in journal (Refereed) Published
Abstract [en]

The bacterial ribosome is an important drug target for antibiotics that can inhibit different stages of protein synthesis. Among the various classes of compounds that impair translation there are, however, no known small-molecule inhibitors that specifically target ribosomal release factors (RFs). The class I RFs are essential for correct termination of translation and they differ considerably between bacteria and eukaryotes, making them potential targets for inhibiting bacterial protein synthesis. We carried out virtual screening of a large compound library against 3D structures of free and ribosome-bound RFs in order to search for small molecules that could potentially inhibit termination by binding to the RFs. Here, we report identification of two such compounds which are found both to bind free RFs in solution and to inhibit peptide release on the ribosome, without affecting peptide bond formation.

Place, publisher, year, edition, pages
Nature Publishing Group, 2019
National Category
Medicinal Chemistry Structural Biology
Identifiers
urn:nbn:se:uu:diva-396310 (URN)10.1038/s41598-019-51977-1 (DOI)000492832300009 ()31659219 (PubMedID)
Funder
Swedish Research CouncilKnut and Alice Wallenberg FoundationSwedish National Infrastructure for Computing (SNIC)
Available from: 2019-11-01 Created: 2019-11-01 Last updated: 2019-11-18Bibliographically approved
Kumar, A., Åqvist, J. & Satpati, P. (2019). Principles of tRNA(Ala) Selection by Alanyl-tRNA Synthetase Based on the Critical G3.U70 Base Pair. ACS OMEGA, 4(13), 15539-15548
Open this publication in new window or tab >>Principles of tRNA(Ala) Selection by Alanyl-tRNA Synthetase Based on the Critical G3.U70 Base Pair
2019 (English)In: ACS OMEGA, ISSN 2470-1343, Vol. 4, no 13, p. 15539-15548Article in journal (Refereed) Published
Abstract [en]

Throughout evolution, the presence of a single G3.U70 mismatch in the acceptor stem of tRNA(Ala) is the major determinant for aminoacylation with alanine by alanyl-tRNA synthetase (AlaRS). Recently reported crystal structures of the complexes AlaRS-tRNA(Ala)/G3.U70 and AlaRS-tRNA tRNA(Ala)/A3.U70 suggest two very different conformations, representing a reactive and a nonreactive state, respectively. On the basis of these structures, it has been proposed that the G3.U70 base pair guides the-CCA end of the tRNA acceptor stem into the active site of AIaRS, thereby enabling aminoacylation. The crystal structures open up the possibility of directly computing the energetics of tRNA specificity by AIaRS. We have carried out molecular dynamics free-energy simulations to quantitatively estimate tRNA discrimination by AlaRS, focusing on the mutations of the single critical base pair G3.U70 to uncover the energetics underlying the accuracy of tRNA selection. The calculations show that the reactive complex is highly selective in favor of the cognate tRNA(Ala)/G3.U70 over its noncognate analogues (A3.U70/G3.C70/A3.C70). In contrast, the nonreactive complex is predicted to be unselective between tRNA(Ala)G3.U70 and tRNA(Ala)/A3.U70. Utilizing our calculated relative binding free energies, we show how a simple three-step kinetic scheme for aminoacylation, involving both an initial nonspecific binding step and a subsequent transition to a selective reactive complex, accounts for the observed kinetics of the process.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-396664 (URN)10.1021/acsomega.9b01827 (DOI)000488852700025 ()31572855 (PubMedID)
Funder
Knut and Alice Wallenberg FoundationSwedish Research Council
Available from: 2019-11-12 Created: 2019-11-12 Last updated: 2019-11-12Bibliographically approved
Jespers, W., Esguerra, M., Åqvist, J. & Gutiérrez-de-Terán, H. (2019). QligFEP: an automated workflow for small molecule free energy calculations in Q. Journal of Cheminformatics, 11, Article ID 26.
Open this publication in new window or tab >>QligFEP: an automated workflow for small molecule free energy calculations in Q
2019 (English)In: Journal of Cheminformatics, ISSN 1758-2946, E-ISSN 1758-2946, Vol. 11, article id 26Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
BMC, 2019
Keywords
Free energy perturbation (FEP), Molecular dynamics (MD), Ligand binding, Application programming interface (API), Dual topology
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-382389 (URN)10.1186/s13321-019-0348-5 (DOI)000463589200001 ()30941533 (PubMedID)
Funder
Swedish Research CouncileSSENCE - An eScience Collaboration
Available from: 2019-04-26 Created: 2019-04-26 Last updated: 2019-04-26Bibliographically approved
Jespers, W., Isaksen, G. V., Andberg, T. A., Vasile, S., van Veen, A., Åqvist, J., . . . Gutiérrez-de-Terán, H. (2019). QresFEP: An Automated Protocol for Free Energy Calculations of Protein Mutations in Q. Journal of Chemical Theory and Computation, 15(10), 5461-5473
Open this publication in new window or tab >>QresFEP: An Automated Protocol for Free Energy Calculations of Protein Mutations in Q
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2019 (English)In: Journal of Chemical Theory and Computation, ISSN 1549-9618, E-ISSN 1549-9626, Vol. 15, no 10, p. 5461-5473Article in journal (Refereed) Published
Abstract [en]

Predicting the effect of single-point mutations on protein stability or protein-ligand binding is a major challenge in computational biology. Free energy calculations constitute the most rigorous approach to this problem, though the estimation of converged values for amino acid mutations remains challenging. To overcome this limitation, we developed tailored protocols to calculate free energy shifts associated with single-point mutations. We herein describe the QresFEP protocol, which includes an extension of our recent protocols to cover all amino acids mutations, based on the latest versions of the OPLS-AA force field. QresFEP is implemented in an application programming interface framework and the graphic interface QGui, for the molecular dynamics software Q. The complete protocol is benchmarked in several model systems, optimizing a number of sampling parameters and the implementation of Zwanzig's exponential formula and Bennet's acceptance ratio methods. QresFEP shows an excellent performance on estimating the hydration free energies of amino acid side-chain mimics, including their charged analogues. We also examined its performance on a protein-ligand binding problem of pharmaceutical relevance, the antagonism of neuropeptide Y1 G protein-coupled receptor. Here, the calculations show very good agreement with the experimental effect of 16 mutations on the binding of antagonists BIBP3226, in line with our recent applications in this field. Finally, the characterization of 43 mutations of T4-lysozyme reveals the capacity of our protocol to assess variations of the thermal stability of proteins, achieving a similar performance to alternative free energy perturbation (FEP) approaches. In summary, QresFEP is a robust, versatile, and user-friendly computational FEP protocol to examine biochemical effects of single-point mutations with high accuracy.

National Category
Bioinformatics (Computational Biology)
Identifiers
urn:nbn:se:uu:diva-395642 (URN)10.1021/acs.jctc.9b00538 (DOI)000489678700026 ()31436990 (PubMedID)
Funder
The Research Council of Norway, 262695 274858Swedish Research CouncilKnut and Alice Wallenberg FoundationeSSENCE - An eScience Collaboration
Available from: 2019-10-22 Created: 2019-10-22 Last updated: 2019-11-06Bibliographically approved
Socan, J., Isaksen, G. V., Brandsdal, B. O. & Åqvist, J. (2019). Towards Rational Computational Engineering of Psychrophilic Enzymes. Scientific Reports, 9, Article ID 19147.
Open this publication in new window or tab >>Towards Rational Computational Engineering of Psychrophilic Enzymes
2019 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 19147Article in journal (Refereed) Published
Abstract [en]

Cold-adapted enzymes from psychrophilic species achieve their high catalytic efficiency at low temperature by a different partitioning of the activation free energy into its enthalpic and entropic components, compared to orthologous mesophilic enzymes. Their lower activation enthalpy, partly compensated by an increased entropic penalty, has been suggested to originate from changes in flexibility of the protein surface. Multiple sequence alignments of psychrophilic and mesophilic enzymes also show characteristic motifs located in surface loops of the protein. Here, we use computer simulations to examine the effects of a number of designed surface mutations of psychrophilic and mesophilic elastases on the temperature dependence of the catalyzed peptide cleavage reaction. For each of 14 mutant enzyme variants we report calculations of their thermodynamic activation parameters. The results show that substitution of psychrophilic loop residues into the mesophilic enzyme consistently changes both the activation parameters and loop flexibilities towards the former, and vice versa for opposite substitutions.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2019
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-402233 (URN)10.1038/s41598-019-55697-4 (DOI)000503186000003 ()31844096 (PubMedID)
Funder
Swedish Research CouncilKnut and Alice Wallenberg FoundationThe Research Council of Norway
Available from: 2020-01-16 Created: 2020-01-16 Last updated: 2020-01-16Bibliographically approved
Socan, J., Kazemi, M., Isaksen, G. V., Brandsdal, B. O. & Åqvist, J. (2018). Catalytic Adaptation of Psychrophilic Elastase. Biochemistry, 57(20), 2984-2993
Open this publication in new window or tab >>Catalytic Adaptation of Psychrophilic Elastase
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2018 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 57, no 20, p. 2984-2993Article in journal (Refereed) Published
Abstract [en]

The class I pancreatic elastase from Atlantic salmon is considered to be a cold-adapted enzyme in view of the cold habitat, the reduced thermostability of the enzyme, and the fact that it is faster than its mesophilic porcine counterpart at room temperature. However, no experimental characterization of its catalytic properties at lower temperatures has actually been reported. Here we use extensive computer simulations of its catalytic reaction, at different temperatures and with different peptide substrates, to compare its characteristics with those of porcine pancreatic elastase, with which it shares 67% sequence identity. We find that both enzymes have a preference for smaller aliphatic residues at the P1 position, while the reaction rate with phenylalanine at P1 is predicted to be substantially lower. With the former class of substrates, the calculated reaction rates for salmon enzyme are consistently higher than those of the porcine ortholog at all temperatures examined, and the difference is most pronounced at the lowest temperature. As observed for other cold-adapted enzymes, this is caused by redistribution of the activation free energy in terms of enthalpy and entropy and can be linked to differences in the mobility of surface-exposed loops in the two enzymes. Such mobility changes are found to be reflected by characteristic sequence conservation patterns in psychrophilic and mesophilic species. Hence, calculations of mutations in a single surface loop show that the temperature dependence of the catalytic reaction is altered in a predictable way.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-357569 (URN)10.1021/acs.biochem.8b00078 (DOI)000433404600009 ()29726678 (PubMedID)
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2018-08-17 Created: 2018-08-17 Last updated: 2018-08-17Bibliographically approved
Vasile, S., Esguerra, M., Jespers, W., Oliveira, A., Sallander, J., Åqvist, J. & Gutiérrez-de-Terán, H. (2018). Characterization of Ligand Binding to GPCRs Through Computational Methods.. In: Computational Methods for GPCR Drug Discovery: (pp. 23-44). New York, NY: Humana Press, 1705
Open this publication in new window or tab >>Characterization of Ligand Binding to GPCRs Through Computational Methods.
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2018 (English)In: Computational Methods for GPCR Drug Discovery, New York, NY: Humana Press, 2018, Vol. 1705, p. 23-44Chapter in book (Refereed)
Abstract [en]

The recent increase in available G protein-coupled receptor structures now contributes decisively to the structure-based ligand design. In this context, computational approaches in combination with medicinal chemistry and pharmacology are extremely helpful. Here, we provide an update on our structure-based computational protocols, used to answer key questions related to GPCR-ligand binding. All combined, these techniques can shed light on ligand binding modes, determine the molecular basis of conformational selection, for agonists and antagonists, as well as of subtype selectivity. To illustrate each of these questions, we will consider examples from existing projects on three families of class A (rhodopsin-like) GPCRs: one small-molecule (nucleotide-like) family, i.e., the adenosine receptors, and two peptide-binding receptors: neuropeptide-Y and angiotensin II receptors. The successful application of the same computational protocols to investigate this diverse group of receptor families gives an idea of the general applicability of our methodology in the characterization of GPCR-ligand binding.

Place, publisher, year, edition, pages
New York, NY: Humana Press, 2018
Keywords
Free energy perturbation, Homology modeling, Molecular dynamics, Structure-based drug design
National Category
Bioinformatics (Computational Biology)
Identifiers
urn:nbn:se:uu:diva-395745 (URN)10.1007/978-1-4939-7465-8_2 (DOI)29188557 (PubMedID)
Available from: 2019-10-23 Created: 2019-10-23 Last updated: 2020-01-22Bibliographically approved
Ge, X., Mandava, C. S., Lind, C., Åqvist, J. & Sanyal, S. (2018). Complementary charge-based interaction between the ribosomal-stalk protein L7/12 and IF2 is the key to rapid subunit association. Proceedings of the National Academy of Sciences of the United States of America, 115(18), 4649-4654
Open this publication in new window or tab >>Complementary charge-based interaction between the ribosomal-stalk protein L7/12 and IF2 is the key to rapid subunit association
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2018 (English)In: 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) Published
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.

Keywords
protein synthesis, ribosomal protein L7/12, protein-protein interaction, ribosome, translation initiation
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-350185 (URN)10.1073/pnas.1802001115 (DOI)000431119600050 ()29686090 (PubMedID)
Funder
Swedish Research Council, 2014-4423; 2016-06264Knut and Alice Wallenberg Foundation, 2011.0081VINNOVA, 2013-8778
Available from: 2018-05-07 Created: 2018-05-07 Last updated: 2018-07-13Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-2091-0610

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