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  • 1.
    Borroto-Escuela, Dasiel O.
    et al.
    Karolinska Inst, Dept Neurosci, Stockholm, Sweden.;Univ Urbino, Physiol Sect, Dept Biomol Sci, Urbino, Italy.;Grp Bohio Estudio, Observ Cubano Neurociencias, Yaguajay, Cuba..
    Carlsson, Jens
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Ambrogini, Patricia
    Univ Urbino, Physiol Sect, Dept Biomol Sci, Urbino, Italy..
    Narvaez, Manuel
    Univ Malaga, Inst Invest Biomed Malaga, Fac Med, Malaga, Spain..
    Wydra, Karolina
    Polish Acad Sci, Inst Pharmacol, Dept Pharmacol, Labo Drug Addict Pharmacol, Krakow, Poland..
    Tarakanov, Alexander O.
    Russian Acad Sci, St Petersburg Inst Informat & Automat, St Petersburg, Russia..
    Li, Xiang
    Karolinska Inst, Dept Neurosci, Stockholm, Sweden..
    Millon, Carmelo
    Univ Malaga, Inst Invest Biomed Malaga, Fac Med, Malaga, Spain..
    Ferraro, Luca
    Univ Ferrara, Dept Life Sci & Biotechnol, Ferrara, Italy..
    Cuppini, Riccardo
    Univ Urbino, Physiol Sect, Dept Biomol Sci, Urbino, Italy..
    Tanganelli, Sergio
    Univ Ferrara, Dept Med Sci, Ferrara, Italy..
    Liu, Fang
    Univ Toronto, Ctr Addict & Mental Hlth, Campbell Res Inst, Toronto, ON, Canada..
    Filip, Malgorzata
    Laboratory of Drug Addiction Pharmacology, Department of Pharmacology, Institute of Pharmacology, Polish Academy of Sciences, Kraków, Poland.
    Diaz-Cabiale, Zaida
    Univ Malaga, Inst Invest Biomed Malaga, Fac Med, Malaga, Spain..
    Fuxe, Kjell
    Karolinska Inst, Dept Neurosci, Stockholm, Sweden..
    Understanding the Role of GPCR Heteroreceptor Complexes in Modulating the Brain Networks in Health and Disease2017In: Frontiers in Cellular Neuroscience, ISSN 1662-5102, E-ISSN 1662-5102, Vol. 11, article id 37Article, review/survey (Refereed)
    Abstract [en]

    The introduction of allosteric receptor-receptor interactions in G protein-coupled receptor (GPCR) heteroreceptor complexes of the central nervous system (CNS) gave a new dimension to brain integration and neuropsychopharmacology. The molecular basis of learning and memory was proposed to be based on the reorganization of the homo- and heteroreceptor complexes in the postjunctional membrane of synapses. Long-term memory may be created by the transformation of parts of the heteroreceptor complexes into unique transcription factors which can lead to the formation of specific adapter proteins. The observation of the GPCR heterodimer network (GPCR-HetNet) indicated that the allosteric receptor-receptor interactions dramatically increase GPCR diversity and biased recognition and signaling leading to enhanced specificity in signaling. Dysfunction of the GPCR heteroreceptor complexes can lead to brain disease. The findings of serotonin (5-HT) hetero and isoreceptor complexes in the brain over the last decade give new targets for drug development in major depression. Neuromodulation of neuronal networks in depression via 5-HT, galanin peptides and zinc involve a number of GPCR heteroreceptor complexes in the raphe-hippocampal system: GalR1-5-HT1A, GalR1-5-HT1A-GPR39, GalR1-GalR2, and putative GalR1-GalR2-5-HT1A heteroreceptor complexes. The 5-HT1A receptor protomer remains a receptor enhancing antidepressant actions through its participation in hetero- and homoreceptor complexes listed above in balance with each other. In depression, neuromodulation of neuronal networks in the raphe-hippocampal system and the cortical regions via 5-HT and fibroblast growth factor 2 involves either FGFR1-5-HT1A heteroreceptor complexes or the 5-HT isoreceptor complexes such as 5-HT1A-5-HT7 and 5-HT1A-5-HT2A. Neuromodulation of neuronal networks in cocaine use disorder via dopamine (DA) and adenosine signals involve A2AR-D2R and A2AR-D2R-Sigma1R heteroreceptor complexes in the dorsal and ventral striatum. The excitatory modulation by A2AR agonists of the ventral striato-pallidal GABA anti-reward system via targeting the A2AR-D2R and A2AR-D2R-Sigma1R heteroreceptor complex holds high promise as a new way to treat cocaine use disorders. Neuromodulation of neuronal networks in schizophrenia via DA, adenosine, glutamate, 5-HT and neurotensin peptides and oxytocin, involving A2AR-D2R, D2R-NMDAR, A2AR-D2R-mGluR5, D2R-5-HT2A and D2R-oxytocinR heteroreceptor complexes opens up a new world of D2R protomer targets in the listed heterocomplexes for treatment of positive, negative and cognitive symptoms of schizophrenia.

  • 2.
    Borroto-Escuela, Dasiel O.
    et al.
    Karolinska Inst, Dept Neurosci, Stockholm, Sweden.
    Rodriguez, David
    Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, Stockholm, Sweden.
    Romero Fernandez, Wilber
    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.
    Kapla, Jon
    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.
    Jaiteh, Mariama
    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.
    Ranganathan, Anirudh
    Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, Stockholm, Sweden.
    Lazarova, Tzvetana
    Autonomous Univ Barcelona, Fac Med, Dept Biochem & Mol Biol, Inst Neurosci, Barcelona, Spain.
    Fuxe, Kjell
    Karolinska Inst, Dept Neurosci, Stockholm, Sweden.
    Carlsson, Jens
    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.
    Mapping the Interface of a GPCR Dimer: A Structural Model of the A(2A) Adenosine and D-2 Dopamine Receptor Heteromer2018In: Frontiers in Pharmacology, ISSN 1663-9812, E-ISSN 1663-9812, Vol. 9, article id 829Article in journal (Refereed)
    Abstract [en]

    The A(2A) adenosine (A(2A)R) and D-2 dopamine (D2R) receptors form oligomers in the cell membrane and allosteric interactions across the A(2A)R-D2R heteromer represent a target for development of drugs against central nervous system disorders. However, understanding of the molecular determinants of A(2A)R-D2R heteromerization and the allosteric antagonistic interactions between the receptor protomers is still limited. In this work, a structural model of the A(2A)R-D2R heterodimer was generated using a combined experimental and computational approach. Regions involved in the heteromer interface were modeled based on the effects of peptides derived from the transmembrane (TM) helices on A(2A)R-D2R receptor-receptor interactions in bioluminescence resonance energy transfer (BRET) and proximity ligation assays. Peptides corresponding to TM-IV and TM-V of the A(2A)R blocked heterodimer interactions and disrupted the allosteric effect of A(2A)R activation on D2R agonist binding. Protein-protein docking was used to construct a model of the A(2A)R-D2R heterodimer with a TM-IV/V interface, which was refined using molecular dynamics simulations. Mutations in the predicted interface reduced A(2A)R-D2R interactions in BRET experiments and altered the allosteric modulation. The heterodimer model provided insights into the structural basis of allosteric modulation and the technique developed to characterize the A(2A)R-D2R interface can be extended to study the many other G protein-coupled receptors that engage in heteroreceptor complexes.

  • 3.
    Borroto-Escuela, Dasiel O.
    et al.
    Univ Urbino, Dept Biomol Sci, Sect Physiol, Campus Sci Enrico Mattei,Via Ca le Suore 2, I-61029 Urbino, Italy;Grp Bohio Estudio, Observ Cubano Neurociencias, Zayas 50, Yaguajay 62100, Cuba;Karolinska Inst, Dept Neurosci, Retzius Vag 8, S-17177 Stockholm, Sweden.
    Wydra, Karolina
    Polish Acad Sci, Inst Pharmacol, Dept Drug Addict Pharmacol, 12 Smetna St, PL-31343 Krakow, Poland.
    Li, Xiang
    Jilin Univ, Coll Life Sci, Qianjin St 2699, Changchun 130012, Jilin, Peoples R China;Karolinska Inst, Dept Neurosci, Retzius Vag 8, S-17177 Stockholm, Sweden.
    Rodriguez, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab. Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, SE-10691 Stockholm, Sweden.
    Carlsson, Jens
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab. Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, SE-10691 Stockholm, Sweden.
    Jastrzebska, Joanna
    Polish Acad Sci, Inst Pharmacol, Dept Drug Addict Pharmacol, 12 Smetna St, PL-31343 Krakow, Poland.
    Filip, Malgorzata
    Polish Acad Sci, Inst Pharmacol, Dept Drug Addict Pharmacol, 12 Smetna St, PL-31343 Krakow, Poland.
    Fuxe, Kjell
    Karolinska Inst, Dept Neurosci, Retzius Vag 8, S-17177 Stockholm, Sweden.
    Disruption of A2AR-D2R Heteroreceptor Complexes After A2AR Transmembrane 5 Peptide Administration Enhances Cocaine Self-Administration in Rats2018In: Molecular Neurobiology, ISSN 0893-7648, E-ISSN 1559-1182, Vol. 55, no 8, p. 7038-7048Article in journal (Refereed)
    Abstract [en]

    Antagonistic allosteric A2AR-D2R receptor-receptor interactions in heteroreceptor complexes counteract cocaine self-administration and cocaine seeking in rats as seen in biochemical and behavioral experiments. It was shown that the human A2AR transmembrane five (TM5) was part of the interface of the human A2AR-D2R receptor heteromer. In the current paper, the rat A2AR synthetic TM5 (synthTM5) peptide disrupts the A2AR-D2R heteroreceptor complex in HEK293 cells as shown by the bioluminescence resonance energy transfer method. Rat A2AR synthTM5 peptide, microinjected into the nucleus accumbens, produced a complete counteraction of the inhibitory effects of the A2AR agonist CGS21680 on cocaine self-administration. It was linked to a disappearance of the accumbal A2AR-D2R heteroreceptor complexes and the A2AR agonist induced inhibition of D2R recognition using proximity ligation assay and biochemical binding techniques. However, possible effects of the A2AR synthTM5 peptide on accumbal A2AR-D3R and A2AR-D4R heteroreceptor complexes remain to be excluded. Evidence is provided that accumbal A2AR-D2R-like heteroreceptor complexes with their antagonistic receptor-receptor interactions can be major targets for treatment of cocaine use disorder.

  • 4.
    Carlsson, Jens
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Structure-based screening for GPCR ligands from fragment and lead-like chemical space2018In: Abstract of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 255Article in journal (Other academic)
  • 5.
    Carlsson, Jens
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Structure-guided discovery of adenosine receptor ligands2018In: Purinergic Signalling Purinergic Signalling, ISSN 1573-9538, E-ISSN 1573-9546, Vol. 14, no Suppl. 1, p. S51-S51Article in journal (Other academic)
  • 6.
    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.

  • 7.
    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.

  • 8.
    Jaiteh, Mariama
    et al.
    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.
    Zeifman, Alexey
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Saarinen, Marcus
    Karolinska Inst, Dept Physiol & Pharmacol, Ctr Mol Med, SE-17177 Stockholm, Sweden.
    Svenningsson, Per
    Karolinska Inst, Dept Physiol & Pharmacol, Ctr Mol Med, SE-17177 Stockholm, Sweden.
    Brea, Jose
    Univ Santiago de Compostela, Ctr Res Mol Med & Chron Dis, USEF Screening Platform BioFarma Res Grp, Santiago De Compostela 15706, Spain.
    Loza, Maria Isabel
    Univ Santiago de Compostela, Ctr Res Mol Med & Chron Dis, USEF Screening Platform BioFarma Res Grp, Santiago De Compostela 15706, Spain.
    Carlsson, Jens
    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. Uppsala Univ, Dept Cell & Mol Biol, Sci Life Lab, BMC Box 596, SE-75124 Uppsala, Sweden.
    Docking Screens for Dual Inhibitors of Disparate Drug Targets for Parkinson's Disease2018In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 61, no 12, p. 5269-5278Article in journal (Refereed)
    Abstract [en]

    Modulation of multiple biological targets with a single drug can lead to synergistic therapeutic effects and has been demonstrated to be essential for efficient treatment of CNS disorders. However, rational design of compounds that interact with several targets is very challenging. Here, we demonstrate that structure-based virtual screening can guide the discovery of multi-target ligands of unrelated proteins relevant for Parkinson's disease. A library with 5.4 million molecules was docked to crystal structures of the A(2A) adenosine receptor (A(2A)AR) and monoamine oxidase B (MAO-B). Twenty-four compounds that were among the highest ranked for both binding sites were evaluated experimentally, resulting in the discovery of four dual-target ligands. The most potent compound was an A(2A)AR antagonist with nanomolar affinity (K-i = 19 nM) and inhibited MAO-B with an IC50 of 100 nM. Optimization guided by the predicted binding modes led to the identification of a second potent dual-target scaffold. The two discovered scaffolds were shown to counteract 6-hydroxydopamine-induced neurotoxicity in dopaminergic neuronal-like SH-SY5Y cells. Structure-based screening can hence be used to identify ligands with specific polypharmacological profiles, providing new avenues for drug development against complex diseases.

  • 9.
    Kennedy, Amanda
    et al.
    Discovery Sciences, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden.
    Ballante, Flavio
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Johansson, Johan
    Cardiovascular Renal and Metabolism, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden.
    Milligan, Graeme
    Centre for Translational Pharmacology, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow.
    Sundström, Linda
    Discovery Sciences, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden.
    Nordqvist, Anneli
    Cardiovascular Renal and Metabolism, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden.
    Carlsson, Jens
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Structural Characterization of Agonist Binding to Protease-Activated Receptor 2 through Mutagenesis and Computational Modeling2018In: ACS Pharmacology & Translational Science, ISSN 2575-9108, Vol. 1, no 2, p. 119-133Article in journal (Refereed)
    Abstract [en]

    Protease-activated receptor 2 (PAR2) is a G protein-coupled receptor that is activated by proteolytic cleavage of its N-terminus. The unmasked N-terminal peptide then binds to the transmembrane bundle, leading to activation of intracellular signaling pathways associated with inflammation and cancer. Recently determined crystal structures have revealed binding sites of PAR2 antagonists, but the binding mode of the peptide agonist remains unknown. In order to generate a model of PAR2 in complex with peptide SLIGKV, corresponding to the trypsin-exposed tethered ligand, the orthosteric binding site was probed by iterative combinations of receptor mutagenesis, agonist ligand modifications and data-driven structural modeling. Flexible-receptor docking identified a conserved binding mode for agonists related to the endogenous ligand that was consistent with the experimental data and allowed synthesis of a novel peptide (1-benzyl-1H[1,2,3]triazole-4-yl-LIGKV) with higher functional potency than SLIGKV. The final model may be used to understand the structural basis of PAR2 activation and in virtual screens to identify novel PAR2 agonist and competitive antagonists. The combined experimental and computational approach to characterize agonist binding to PAR2 can be extended to study the many other G protein-coupled receptors that recognize peptides or proteins.

  • 10. Lam, V. M.
    et al.
    Rodriguez, D.
    Zhang, T.
    Koh, E. J.
    Carlsson, Jens
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Salahpour, A.
    Discovery of trace amine-associated receptor 1 ligands by molecular docking screening against a homology model2015In: MedChemComm, ISSN 2040-2503, E-ISSN 2040-2511, Vol. 6, p. 2216-2223Article in journal (Refereed)
    Abstract [en]

    Trace Amines (TA) are side-products of the synthesis of classical neurotransmitters within the brain. TAs exert their effect by binding to a family of G protein-coupled receptors termed Trace Amine-Associated Receptors (TAARs). TAAR1 is the best characterised member of this family and studies on TAAR1 have shown that this receptor is a negative regulator of dopamine transmission. Considering the limited number of pharmacological probes available for TAAR1, we aimed to identify novel ligands of this receptor using structure-based virtual screening. A homology model of TAAR1 was generated and over three million commercially available compounds were screened against the orthosteric site using molecular docking. Among the 42 top-ranked compounds that were tested in functional assays, three partial agonists with EC50 values ranging from 1 to 52 [small mu ]M were discovered. In addition, four potentially weak antagonists were identified. Ten analogs of the two most potent agonists from the screen were also evaluated and three of these displayed equal or greater activity compared to the parent compound. Several of the discovered ligands represent novel scaffolds and are thus promising starting points for development of new pharmacological tools for studying TAAR1 biology.

  • 11.
    Lundgren, Camilla A. K.
    et al.
    Stockholm Univ, Dept Biochem & Biophys, Stockholm, Sweden.
    Sjostrand, Dan
    Stockholm Univ, Dept Biochem & Biophys, Stockholm, Sweden.
    Biner, Olivier
    Univ Bern, Dept Chem & Biochem, Bern, Switzerland.
    Bennett, Matthew
    Stockholm Univ, Dept Biochem & Biophys, Stockholm, Sweden.
    Rudling, Axel
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Johansson, Ann-Louise
    Stockholm Univ, Dept Biochem & Biophys, Stockholm, Sweden.
    Brzezinski, Peter
    Stockholm Univ, Dept Biochem & Biophys, Stockholm, Sweden.
    Carlsson, Jens
    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.
    von Ballmoos, Christoph
    Univ Bern, Dept Chem & Biochem, Bern, Switzerland.
    Högbom, Martin
    Stockholm Univ, Dept Biochem & Biophys, Stockholm, Sweden.
    Scavenging of superoxide by a membrane-bound superoxide oxidase2018In: Nature Chemical Biology, ISSN 1552-4450, E-ISSN 1552-4469, Vol. 14, no 8, p. 788-793Article in journal (Refereed)
    Abstract [en]

    Superoxide is a reactive oxygen species produced during aerobic metabolism in mitochondria and prokaryotes. It causes damage to lipids, proteins and DNA and is implicated in cancer, cardiovascular disease, neurodegenerative disorders and aging. As protection, cells express soluble superoxide dismutases, disproportionating superoxide to oxygen and hydrogen peroxide. Here, we describe a membrane-bound enzyme that directly oxidizes superoxide and funnels the sequestered electrons to ubiquinone in a diffusion-limited reaction. Experiments in proteoliposomes and inverted membranes show that the protein is capable of efficiently quenching superoxide generated at the membrane in vitro. The 2.0 angstrom crystal structure shows an integral membrane di-heme cytochrome b poised for electron transfer from the P-side and proton uptake from the N-side. This suggests that the reaction is electrogenic and contributes to the membrane potential while also conserving energy by reducing the quinone pool. Based on this enzymatic activity, we propose that the enzyme family be denoted superoxide oxidase (SOO).

  • 12.
    Mannel, Barbara
    et al.
    Friedrich Alexander Univ, Dept Chem & Pharm, Med Chem, Schuhstr 19, D-91052 Erlangen, Germany..
    Jaiteh, Mariama
    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.
    Zeifman, Alexey
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Randakova, Alena
    Friedrich Alexander Univ, Dept Chem & Pharm, Med Chem, Schuhstr 19, D-91052 Erlangen, Germany..
    Moller, Dorothee
    Friedrich Alexander Univ, Dept Chem & Pharm, Med Chem, Schuhstr 19, D-91052 Erlangen, Germany..
    Hubher, Harald
    Friedrich Alexander Univ, Dept Chem & Pharm, Med Chem, Schuhstr 19, D-91052 Erlangen, Germany..
    Gmeiner, Peter
    Friedrich Alexander Univ, Dept Chem & Pharm, Med Chem, Schuhstr 19, D-91052 Erlangen, Germany..
    Carlsson, Jens
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Structure-Guided Screening for Functionally Selective D-2 Dopamine Receptor Ligands from a Virtual Chemical Library2017In: ACS Chemical Biology, ISSN 1554-8929, E-ISSN 1554-8937, Vol. 12, no 10, p. 2652-2661Article in journal (Refereed)
    Abstract [en]

    Functionally selective ligands stabilize conformations of G protein-coupled receptors (GPCRs) that induce a preference for signaling via a subset of the intracellular pathways activated by the endogenous agonists. The possibility to fine-tune the functional activity of a receptor provides opportunities to develop drugs that selectively signal via pathways associated with a therapeutic effect and avoid those causing side effects. Animal studies have indicated that ligands displaying functional selectivity at the D-2 dopamine receptor (D2R) could be safer and more efficacious drugs against neuropsychiatric diseases. In this work, computational design of functionally selective D2R ligands was explored using structure-based virtual screening. Molecular docking of known functionally selective ligands to a D2R homology model indicated that such compounds were anchored by interactions with the orthosteric site and extended into a common secondary pocket. A tailored virtual library with close to 13-000 compounds bearing 2,3-dichlorophenylpiperazine, a privileged orthosteric scaffold, connected to diverse chemical moieties via a linker was docked to the D2R model. Eighteen top-ranked compounds that occupied both the orthosteric and allosteric site were synthesized, leading to the discovery of 16 partial agonists. A majority of the ligands had comparable maximum effects in the G protein and beta-arrestin recruitment assays, but a subset displayed preference for a single pathway. In particular, compound 4 stimulated beta-arrestin recruitment (EC50 = 320 nM, E-max = 16%) but had no detectable G protein signaling. The use of structure-based screening and virtual libraries to discover GPCR ligands with tailored functional properties will be discussed.

  • 13.
    Matricon, Pierre
    et al.
    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.
    Ranganathan, Anirudh
    Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, SE-10691 Stockholm, Sweden..
    Warnick, Eugene
    NIDDK, Mol Recognit Sect, Bioorgan Chem Lab, NIH, Bethesda, MD 20892 USA..
    Gao, Zhan-Guo
    NIDDK, Mol Recognit Sect, Bioorgan Chem Lab, NIH, Bethesda, MD 20892 USA..
    Rudling, Axel
    Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, SE-10691 Stockholm, Sweden..
    Lambertucci, Catia
    Univ Camerino, Scuola Sci Farmaco & Prod Salute, Via S Agostino 1, I-62032 Camerino, MC, Italy..
    Marucci, Gabriella
    Univ Camerino, Scuola Sci Farmaco & Prod Salute, Via S Agostino 1, I-62032 Camerino, MC, Italy..
    Ezzati, Aitakin
    Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, SE-10691 Stockholm, Sweden..
    Jaiteh, Mariama
    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.
    Dal Ben, Diego
    Univ Camerino, Scuola Sci Farmaco & Prod Salute, Via S Agostino 1, I-62032 Camerino, MC, Italy..
    Jacobson, Kenneth A.
    NIDDK, Mol Recognit Sect, Bioorgan Chem Lab, NIH, Bethesda, MD 20892 USA..
    Carlsson, Jens
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Fragment optimization for GPCRs by molecular dynamics free energy calculations: Probing druggable subpockets of the A(2A) adenosine receptor binding site2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 6398Article in journal (Refereed)
    Abstract [en]

    Fragment-based lead discovery is becoming an increasingly popular strategy for drug discovery. Fragment screening identifies weakly binding compounds that require optimization to become high-affinity leads. As design of leads from fragments is challenging, reliable computational methods to guide optimization would be invaluable. We evaluated using molecular dynamics simulations and the free energy perturbation method (MD/FEP) in fragment optimization for the A(2A) adenosine receptor, a pharmaceutically relevant G protein-coupled receptor. Optimization of fragments exploring two binding site subpockets was probed by calculating relative binding affinities for 23 adenine derivatives, resulting in strong agreement with experimental data (R-2 = 0.78). The predictive power of MD/FEP was significantly better than that of an empirical scoring function. We also demonstrated the potential of the MD/FEP to assess multiple binding modes and to tailor the thermodynamic profile of ligands during optimization. Finally, MD/FEP was applied prospectively to optimize three nonpurine fragments, and predictions for 12 compounds were evaluated experimentally. The direction of the change in binding affinity was correctly predicted in a majority of the cases, and agreement with experiment could be improved with rigorous parameter derivation. The results suggest that MD/FEP will become a powerful tool in structure-driven optimization of fragments to lead candidates.

  • 14.
    Petersen, Julian
    et al.
    Karolinska Inst, Sect Receptor Biol & Signaling, Dept Physiol & Pharmacol, S-17177 Stockholm, Sweden..
    Wright, Shane C.
    Karolinska Inst, Sect Receptor Biol & Signaling, Dept Physiol & Pharmacol, S-17177 Stockholm, Sweden..
    Rodriguez, David
    Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, SE-10691 Stockholm, Sweden..
    Matricon, Pierre
    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.
    Lahav, Noa
    Hebrew Univ Jerusalem, Inst Chem, IL-91904 Jerusalem, Israel..
    Vromen, Aviv
    Hebrew Univ Jerusalem, Inst Chem, IL-91904 Jerusalem, Israel..
    Friedler, Assaf
    Hebrew Univ Jerusalem, Inst Chem, IL-91904 Jerusalem, Israel..
    Stromqvist, Johan
    Single Technol AB, SE-11428 Stockholm, Sweden..
    Wennmalm, Stefan
    Royal Inst Technol, Dept Appl Phys, Expt Biomol Phys Grp, Sci Life Lab, SE-17165 Solna, Sweden..
    Carlsson, Jens
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Schulte, Gunnar
    Karolinska Inst, Sect Receptor Biol & Signaling, Dept Physiol & Pharmacol, S-17177 Stockholm, Sweden.;Masaryk Univ, Dept Expt Biol, Fac Sci, Kotlarska 2, CS-61137 Brno, Czech Republic..
    Agonist-induced dimer dissociation as a macromolecular step in G protein-coupled receptor signaling2017In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 8, article id 226Article in journal (Refereed)
    Abstract [en]

    G protein-coupled receptors (GPCRs) constitute the largest family of cell surface receptors. They can exist and act as dimers, but the requirement of dimers for agonist-induced signal initiation and structural dynamics remains largely unknown. Frizzled 6 (FZD6) is a member of Class F GPCRs, which bind WNT proteins to initiate signaling. Here, we show that FZD6 dimerizes and that the dimer interface of FZD6 is formed by the transmembrane a-helices four and five. Most importantly, we present the agonist-induced dissociation/re-association of a GPCR dimer through the use of live cell imaging techniques. Further analysis of a dimerization-impaired FZD6 mutant indicates that dimer dissociation is an integral part of FZD6 signaling to extracellular signal-regulated kinases1/2. The discovery of agonistdependent dynamics of dimers as an intrinsic process of receptor activation extends our understanding of Class F and other dimerizing GPCRs, offering novel targets for dimerinterfering small molecules.

  • 15.
    Ranganathan, Anirudh
    et al.
    Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, SE-10691 Stockholm, Sweden..
    Heine, Philipp
    Univ Zurich, Dept Biochem, Winterthurerstr 190, CH-8057 Zurich, Switzerland..
    Rudling, Axel
    Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, SE-10691 Stockholm, Sweden..
    Pluckthun, Andreas
    Univ Zurich, Dept Biochem, Winterthurerstr 190, CH-8057 Zurich, Switzerland..
    Kummer, Lutz
    Univ Zurich, Dept Biochem, Winterthurerstr 190, CH-8057 Zurich, Switzerland.;G7 Therapeut AG, Grabenstr 11a, CH-8952 Schlieren, Switzerland..
    Carlsson, Jens
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Ligand Discovery for a Peptide-Binding GPCR by Structure-Based Screening of Fragment- and Lead-Like Chemical Libraries2017In: ACS Chemical Biology, ISSN 1554-8929, E-ISSN 1554-8937, Vol. 12, no 3, p. 735-745Article in journal (Refereed)
    Abstract [en]

    Peptide-recognizing G protein-coupled receptors (GPCRs) are promising therapeutic targets but often resist drug discovery efforts. Determination of crystal structures for peptide binding GPCRs has provided opportunities to explore structure based methods in lead development. Molecular docking screens of two chemical libraries, containing either fragment- or lead-like compounds, against a neurotensin receptor 1 crystal structure allowed for a comparison between different drug development strategies for peptide-binding GPCRs. A total of 2.3 million molecules were screened computationally, and 25 fragments and 27 leads that were top-ranked in each library were selected for experimental evaluation. Of these, eight fragments and five leads were confirmed as ligands by surface plasmon resonance. The hit rate for the fragment screen (32%) was thus higher than for the lead-like library (19%), but the affinities of the fragments were similar to 100-fold lower. Both screens returned unique scaffolds and demonstrated that a crystal structure of a stabilized peptide-binding GPCR can guide the discovery of small-molecule agonists. The complementary advantages of exploring fragment- and lead-like chemical space suggest that these strategies should be applied synergistically in structure-based screens against challenging GPCR targets.

  • 16. Ranganathan, Anirudh
    et al.
    Stoddart, Leigh A.
    Hill, Stephen J.
    Carlsson, Jens
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Fragment-Based Discovery of Subtype-Selective Adenosine Receptor Ligands from Homology Models2015In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 58, no 24, p. 9578-9590Article in journal (Refereed)
    Abstract [en]

    Fragment-based lead discovery (FBLD) holds great promise for drug discovery, but applications to G protein-coupled receptors (GPCRs) have been limited by a lack of sensitive screening techniques and scarce structural information. If virtual screening against homology models of GPCRs could be used to identify fragment ligands, FBLD could be extended to numerous important drug targets and contribute to efficient lead generation. Access to models of multiple receptors may further enable the discovery of fragments that bind specifically to the desired target. to investigate these questions, we used molecular docking, to screen >500 000 fragments against homology models. of the A(3) and A(1) adenosine receptors (ARs) with the goal to discover,A(3)AR-selective ligands. Twenty-one fragments with predicted A(3)AR-specific binding were evaluated in live-cell fluorescence-based assays; of eight verified ligands, six displayed A(3)/A(1), selectivity,, and three of these had high affinities ranging from 0.1 to 1.3 mu M. Subsequently, structure-guided fragment-to-lead optimization led to the identification of a >100-fold-selective antagonist with nanomolar affinity from commercial libraries. These results highlight that molecular docking screening can guide fragment-based discovery of selective ligands even if the Structures of both the target and antitarget receptors are unknown. The same approach can be readily extended to a large number of pharmaceutically important targets.

  • 17.
    Rodriguez, David
    et al.
    Stockholm Univ, Sci Life Lab, Dept Biochem & Biophys, SE-10691 Stockholm, Sweden.;Stockholm Univ, Ctr Biomembrane Res, SE-10691 Stockholm, Sweden..
    Chakraborty, Saibal
    NIDDK, Mol Recognit Sect, Bioorgan Chem Lab, NIH, Bethesda, MD 20892 USA..
    Warnick, Eugene
    NIDDK, Mol Recognit Sect, Bioorgan Chem Lab, NIH, Bethesda, MD 20892 USA..
    Crane, Steven
    NIDDK, Mol Recognit Sect, Bioorgan Chem Lab, NIH, Bethesda, MD 20892 USA..
    Gao, Zhan-Guo
    NIDDK, Mol Recognit Sect, Bioorgan Chem Lab, NIH, Bethesda, MD 20892 USA..
    O'Connor, Robert
    NIDDK, Mol Recognit Sect, Bioorgan Chem Lab, NIH, Bethesda, MD 20892 USA..
    Jacobson, Kenneth A.
    NIDDK, Mol Recognit Sect, Bioorgan Chem Lab, NIH, Bethesda, MD 20892 USA..
    Carlsson, Jens
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Structure-Based Screening of Uncharted Chemical Space for Atypical Adenosine Receptor Agonists2016In: ACS Chemical Biology, ISSN 1554-8929, E-ISSN 1554-8937, Vol. 11, no 10, p. 2763-2772Article in journal (Refereed)
    Abstract [en]

    Small molecule screening libraries cover only a small fraction of the astronomical number of possible drug-like compounds, limiting the success of ligand discovery efforts. Computational screening of virtual libraries representing unexplored chemical space could potentially bridge this gap. Drug development for adenosine receptors (ARs) as targets for inflammation and cardiovascular diseases has been hampered by the paucity of agonist scaffolds. To identify novel AR agonists, a virtual library of synthetically tractable nucleosides with alternative bases was generated and structure-based virtual screening guided selection of compounds for synthesis. Pharmacological assays were carried out at three AR subtypes for 13 ribosides. Nine compounds displayed significant activity at the ARs, and several of these represented atypical agonist scaffolds. The discovered ligands also provided insights into receptor activation and revealed unknown interactions of endogenous and clinical compounds with the ARs. The results demonstrate that virtual compound databases provide access to bioactive matter from regions of chemical space that are sparsely populated in commercial libraries, an approach transferrable to numerous drug targets.

  • 18.
    Rudling, Axel
    et al.
    Stockholm Univ, Dept Biochem & Biophys, SE-10691 Stockholm, Sweden..
    Gustafsson, Robert
    Stockholm Univ, Dept Biochem & Biophys, SE-10691 Stockholm, Sweden..
    Almlof, Ingrid
    Karolinska Inst, Dept Med Biochem & Biophys, Sci Life Lab, Box 1031, SE-17121 Solna, Sweden..
    Homan, Evert
    Karolinska Inst, Dept Med Biochem & Biophys, Sci Life Lab, Box 1031, SE-17121 Solna, Sweden..
    Scobie, Martin
    Karolinska Inst, Dept Med Biochem & Biophys, Sci Life Lab, Box 1031, SE-17121 Solna, Sweden..
    Berglund, Ulrika Warpman
    Karolinska Inst, Dept Med Biochem & Biophys, Sci Life Lab, Box 1031, SE-17121 Solna, Sweden..
    Helleday, Thomas
    Karolinska Inst, Dept Med Biochem & Biophys, Sci Life Lab, Box 1031, SE-17121 Solna, Sweden..
    Stenmark, Pal
    Stockholm Univ, Dept Biochem & Biophys, SE-10691 Stockholm, Sweden..
    Carlsson, Jens
    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.
    Fragment-Based Discovery and Optimization of Enzyme Inhibitors by Docking of Commercial Chemical Space2017In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 60, no 19, p. 8160-8169Article in journal (Refereed)
    Abstract [en]

    Fragment-based lead discovery has emerged as a leading drug development strategy for novel therapeutic targets. Although fragment-based drug discovery benefits immensely from access to atomic-resolution information, structure-based virtual screening has rarely been used to drive fragment discovery and optimization. Here, molecular docking of 0.3 million fragments to a crystal structure of cancer target MTH1 was performed. Twenty-two predicted fragment ligands, for which analogs could be acquired commercially, were experimentally evaluated. Five fragments inhibited MTH1 with IC50 values ranging from 6 to 79 mu M. Structure-based optimization guided by predicted binding modes and analogs from commercial chemical libraries yielded nanomolar inhibitors. Subsequently solved crystal structures confirmed binding modes predicted by docking for three scaffolds. Structure-guided exploration of commercial chemical space using molecular docking gives access to fragment libraries that are several orders of magnitude larger than those screened experimentally and can enable efficient optimization of hits to potent leads.

  • 19.
    Rudling, Axel
    et al.
    Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, SE-10691 Stockholm, Sweden..
    Orro, Adolfo
    Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, SE-10691 Stockholm, Sweden..
    Carlsson, Jens
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Prediction of Ordered Water Molecules in Protein Binding Sites from Molecular Dynamics Simulations: The Impact of Ligand Binding on Hydration Networks2018In: Journal of Chemical Information and Modeling, ISSN 1549-9596, E-ISSN 1549-960X, Vol. 58, no 2, p. 350-361Article in journal (Refereed)
    Abstract [en]

    Water plays a major role in ligand binding and is attracting increasing attention in structure-based drug design. Water molecules can make large contributions to binding affinity by bridging protein-ligand interactions or by being displaced upon complex formation, but these phenomena are challenging to model at the molecular level. Herein, networks of ordered water molecules in protein binding sites were analyzed by clustering of molecular dynamics (MD) simulation trajectories. Locations of ordered waters (hydration sites) were first identified from simulations of high resolution crystal structures of 13 protein-ligand complexes. The MD-derived hydration sites reproduced 73% of the binding site water molecules observed in the crystal structures. If the simulations were repeated without the cocrystallized ligands, a majority (58%) of the crystal waters in the binding sites were still predicted. In addition, comparison of the hydration sites obtained from simulations carried out in the absence of ligands to those identified for the complexes revealed that the networks of ordered water molecules were preserved to a large extent, suggesting that the locations of waters in a protein-ligand interface are mainly dictated by the protein. Analysis of >1000 crystal structures showed that hydration sites bridged protein-ligand interactions in complexes with different ligands, and those with high MD-derived occupancies were more likely to correspond to experimentally observed ordered water molecules. The results demonstrate that ordered water molecules relevant for modeling of protein-ligand complexes can be identified from MD simulations. Our findings could contribute to development of improved methods for structure-based virtual screening and lead optimization.

  • 20.
    Strakova, Katerina
    et al.
    Masaryk Univ, Fac Sci, Inst Expt Biol, Lab WNT Signaling, Kotiarska 2, Brno 61137, Czech Republic.;Karolinska Inst, Dept Physiol & Pharmacol, Lab Receptor Biol & Signaling, Nanna Svartz Vag 2, S-17177 Stockholm, Sweden..
    Matricon, Pierre
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Yokota, Chika
    Karolinska Inst, Dept Med Biochem & Biophys, Lab Mol Neurobiol, Scheelevag 2, S-17177 Stockholm, Sweden..
    Arthofer, Elisa
    Eunice Kennedy Shriver Natl Inst Child Hlth & Hum, Sect Mol Signal Transduct, NIH, 35A Convent Dr,MSC 3752, Bethesda, MD 20892 USA..
    Bernatik, Ondrej
    Masaryk Univ, Fac Sci, Inst Expt Biol, Lab WNT Signaling, Kotiarska 2, Brno 61137, Czech Republic..
    Rodriguez, David
    Stockholm Univ, Dept Biochem & Biophys, SE-10691 Stockholm, Sweden.;Stockholm Univ, Ctr Biomembrane Res, SE-10691 Stockholm, Sweden..
    Arenas, Ernest
    Karolinska Inst, Dept Med Biochem & Biophys, Lab Mol Neurobiol, Scheelevag 2, S-17177 Stockholm, Sweden..
    Carlsson, Jens
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Bryja, Viterslav
    Masaryk Univ, Fac Sci, Inst Expt Biol, Lab WNT Signaling, Kotiarska 2, Brno 61137, Czech Republic..
    Schulte, Gunnar
    Masaryk Univ, Fac Sci, Inst Expt Biol, Lab WNT Signaling, Kotiarska 2, Brno 61137, Czech Republic.;Karolinska Inst, Dept Physiol & Pharmacol, Lab Receptor Biol & Signaling, Nanna Svartz Vag 2, S-17177 Stockholm, Sweden..
    The tyrosine Y250(2.39) in Frizzled 4 defines a conserved motif important for structural integrity of the receptor and recruitment of Disheveled2017In: Cellular Signalling, ISSN 0898-6568, E-ISSN 1873-3913, Vol. 38, p. 85-96Article in journal (Refereed)
    Abstract [en]

    Frizzleds (FZDs) are unconventional G protein-coupled receptors, which activate diverse intracellular signaling pathways via the phosphoprotein Disheveled (DVL) and heterotrimeric G proteins. The Interaction interplay of FZDs with DVL and G proteins is complex, involves different regions of FZD and the potential dynamics are poorly understood. In the present study, we aimed to characterize the function of a highly conserved tyrosine (Y250(2.39)) in the intracellular loop 1 (ILl) of human FZD(4). We have found Y250(2.39) to be crucial for DVL2 interaction and DVL2 translocation to the plasma membrane. Mutant FZD4-Y250(2.39)F, impaired in DVL2 binding, was defective in both beta-catenin-dependent and beta-catenin-independent WNT signaling induced in Xenopus laevis embryos. The same mutant maintained interaction with the heterotrimeric G proteins Gan and G alpha(13) and was able to mediate WNT-induced G protein dissociation and G protein-dependent YAP/TAZ signaling. We conclude from modeling and dynamics simulation efforts that Y250(2.39) is important for the structural integrity of the FZD-DVL, but not for the FZD-G protein interface and hypothesize that the interaction network of Y250(2.39) and H348(4.46) plays a role in specifying downstream signaling pathways induced by the receptor.

  • 21.
    Strakova, Katerina
    et al.
    Masaryk Univ, Fac Sci, Inst Expt Biol, Lab WNT Signaling, Kotiarska 2, Brno 61137, Czech Republic.;Karolinska Inst, Dept Physiol & Pharmacol, Lab Receptor Biol & Signaling, Nanna Svartz Vag 2, S-17177 Stockholm, Sweden..
    Matricon, Pierre
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Yokota, Chika
    Karolinska Inst, Dept Med Biochem & Biophys, Lab Mol Neurobiol, Scheelevag 2, S-17177 Stockholm, Sweden..
    Arthofer, Elisa
    Eunice Kennedy Shriver Natl Inst Child Hlth & Hum, Sect Mol Signal Transduct, NIH, 35A Convent Dr,MSC 3752, Bethesda, MD 20892 USA..
    Bernatik, Ondrej
    Masaryk Univ, Fac Sci, Inst Expt Biol, Lab WNT Signaling, Kotiarska 2, Brno 61137, Czech Republic..
    Rodriguez, David
    Stockholm Univ, Dept Biochem & Biophys, SE-10691 Stockholm, Sweden.;Stockholm Univ, Ctr Biomembrane Res, SE-10691 Stockholm, Sweden..
    Arenas, Ernest
    Karolinska Inst, Dept Med Biochem & Biophys, Lab Mol Neurobiol, Scheelevag 2, S-17177 Stockholm, Sweden..
    Carlsson, Jens
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Bryja, Viterslav
    Masaryk Univ, Fac Sci, Inst Expt Biol, Lab WNT Signaling, Kotiarska 2, Brno 61137, Czech Republic..
    Schulte, Gunnar
    Masaryk Univ, Fac Sci, Inst Expt Biol, Lab WNT Signaling, Kotiarska 2, Brno 61137, Czech Republic.;Karolinska Inst, Dept Physiol & Pharmacol, Lab Receptor Biol & Signaling, Nanna Svartz Vag 2, S-17177 Stockholm, Sweden..
    The tyrosine Y2502.39 in Frizzled 4 defines a conserved motif important for structural integrity of the receptor and recruitment of Disheveled2017In: Cellular Signalling, ISSN 0898-6568, E-ISSN 1873-3913, Vol. 38, p. 85-96Article in journal (Refereed)
    Abstract [en]

    Frizzleds (FZDs) are unconventional G protein-coupled receptors, which activate diverse intracellular signaling pathways via the phosphoprotein Disheveled (DVL) and heterotrimeric G proteins. The Interaction interplay of FZDs with DVL and G proteins is complex, involves different regions of FZD and the potential dynamics are poorly understood. In the present study, we aimed to characterize the function of a highly conserved tyrosine (Y2502.39) in the intracellular loop 1 (ILl) of human FZD4. We have found Y2502.39 to be crucial for DVL2 interaction and DVL2 translocation to the plasma membrane. Mutant FZD4-Y2502.39F, impaired in DVL2 binding, was defective in both beta-catenin-dependent and beta-catenin-independent WNT signaling induced in Xenopus laevis embryos. The same mutant maintained interaction with the heterotrimeric G proteins Gan and G alpha13 and was able to mediate WNT-induced G protein dissociation and G protein-dependent YAP/TAZ signaling. We conclude from modeling and dynamics simulation efforts that Y2502.39 is important for the structural integrity of the FZD-DVL, but not for the FZD-G protein interface and hypothesize that the interaction network of Y2502.39 and H3484.46 plays a role in specifying downstream signaling pathways induced by the receptor.

  • 22.
    Wright, Shane C.
    et al.
    Karolinska Inst, Sect Receptor Biol & Signaling, Dept Physiol & Pharmacol, S-17165 Stockholm, Sweden;Univ Montreal, Inst Res Immunol & Canc, Dept Biochem & Mol Med, Montreal, PQ H3C 3J7, Canada.
    Canizal, Maria Consuelo Alonso
    Univ Wurzburg, Inst Pharmacol & Toxicol, Versbacher Str 9, D-97078 Wurzburg, Germany;Friedrich Schiller Univ Jena, Univ Hosp Jena, Inst Mol Cell Biol, Ctr Mol Biomed, Hans Knoll Str 2, D-07745 Jena, Germany.
    Benkel, Tobias
    Univ Bonn, Inst Pharmaceut Biol, D-53115 Bonn, Germany.
    Simon, Katharina
    Univ Bonn, Inst Pharmaceut Biol, D-53115 Bonn, Germany.
    Le Gouill, Christian
    Univ Montreal, Inst Res Immunol & Canc, Dept Biochem & Mol Med, Montreal, PQ H3C 3J7, Canada.
    Matricon, Pierre
    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.
    Namkung, Yoon
    McGill Univ, Ctr Hlth, Res Inst, Dept Med, Montreal, PQ H4A 3J1, Canada.
    Lukasheva, Viktoria
    Univ Montreal, Inst Res Immunol & Canc, Dept Biochem & Mol Med, Montreal, PQ H3C 3J7, Canada.
    Koenig, Gabriele M.
    Univ Bonn, Inst Pharmaceut Biol, D-53115 Bonn, Germany.
    Laporte, Stephane A.
    McGill Univ, Ctr Hlth, Res Inst, Dept Med, Montreal, PQ H4A 3J1, Canada;McGill Univ, Dept Pharmacol & Therapeut, Montreal, PQ H3G 1Y6, Canada.
    Carlsson, Jens
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Kostenis, Evi
    Univ Bonn, Inst Pharmaceut Biol, D-53115 Bonn, Germany.
    Bouvier, Michel
    Univ Montreal, Inst Res Immunol & Canc, Dept Biochem & Mol Med, Montreal, PQ H3C 3J7, Canada.
    Schulte, Gunnar
    Karolinska Inst, Sect Receptor Biol & Signaling, Dept Physiol & Pharmacol, S-17165 Stockholm, Sweden.
    Hoffmann, Carsten
    Univ Wurzburg, Inst Pharmacol & Toxicol, Versbacher Str 9, D-97078 Wurzburg, Germany;Friedrich Schiller Univ Jena, Univ Hosp Jena, Inst Mol Cell Biol, Ctr Mol Biomed, Hans Knoll Str 2, D-07745 Jena, Germany.
    FZD(5) is a G alpha(q)-coupled receptor that exhibits the functional hallmarks of prototypical GPCRs2018In: Science Signaling, ISSN 1945-0877, E-ISSN 1937-9145, Vol. 11, no 559, article id eaar5536Article in journal (Refereed)
    Abstract [en]

    Frizzleds (FZDs) are a group of seven transmembrane-spanning (7TM) receptors that belong to class F of the G protein-coupled receptor (GPCR) superfamily. FZDs bind WNT proteins to stimulate diverse signaling cascades involved in embryonic development, stem cell regulation, and adult tissue homeostasis. Frizzled 5 (FZD(5)) is one of the most studied class F GPCRs that promote the functional inactivation of the beta-catenin destruction complex in response to WNTs. However, whether FZDs function as prototypical GPCRs has been heavily debated and, in particular, FZD(5) has not been shown to activate heterotrimeric G proteins. Here, we show that FZD(5) exhibited a conformational change after the addition of WNT-5A, which is reminiscent of class A and class B GPCR activation. In addition, we performed several live-cell imaging and spectrometric-based approaches, such as dual-color fluorescence recovery after photobleaching (dcFRAP) and resonance energy transfer (RET)-based assays that demonstrated that FZD(5) activated G alpha(q) and its downstream effectors upon stimulation with WNT-5A. Together, these findings suggest that FZD(5) is a 7TM receptor with a bona fide GPCR activation profile and suggest novel targets for drug discovery in WNT-FZD signaling.

  • 23.
    Wright, Shane C.
    et al.
    Karolinska Inst, Dept Physiol & Pharmacol, Sect Receptor Biol & Signaling, S-17165 Stockholm, Sweden.
    Kozielewicz, Pawel
    Karolinska Inst, Dept Physiol & Pharmacol, Sect Receptor Biol & Signaling, S-17165 Stockholm, Sweden.
    Kowalski-Jahn, Maria
    Karolinska Inst, Dept Physiol & Pharmacol, Sect Receptor Biol & Signaling, S-17165 Stockholm, Sweden.
    Petersen, Julian
    Karolinska Inst, Dept Physiol & Pharmacol, Sect Receptor Biol & Signaling, S-17165 Stockholm, Sweden.
    Bowin, Carl-Fredrik
    Karolinska Inst, Dept Physiol & Pharmacol, Sect Receptor Biol & Signaling, S-17165 Stockholm, Sweden.
    Slodkowicz, Greg
    MRC Lab Mol Biol, Francis Crick Ave,Cambridge Biomed Campus, Cambridge CB2 0QH, England.
    Marti-Solano, Maria
    MRC Lab Mol Biol, Francis Crick Ave,Cambridge Biomed Campus, Cambridge CB2 0QH, England.
    Rodriguez, David
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Hot, Belma
    Karolinska Inst, Dept Physiol & Pharmacol, Sect Receptor Biol & Signaling, S-17165 Stockholm, Sweden.
    Okashah, Najeah
    Augusta Univ, Med Coll Georgia, Dept Pharmacol & Toxicol, Augusta, GA 30912 USA.
    Strakova, Katerina
    Karolinska Inst, Dept Physiol & Pharmacol, Sect Receptor Biol & Signaling, S-17165 Stockholm, Sweden.
    Valnohova, Jana
    Karolinska Inst, Dept Physiol & Pharmacol, Sect Receptor Biol & Signaling, S-17165 Stockholm, Sweden.
    Babu, M. Madan
    MRC Lab Mol Biol, Francis Crick Ave,Cambridge Biomed Campus, Cambridge CB2 0QH, England.
    Lambert, Nevin A.
    Augusta Univ, Med Coll Georgia, Dept Pharmacol & Toxicol, Augusta, GA 30912 USA.
    Carlsson, Jens
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Schulte, Gunnar
    Karolinska Inst, Dept Physiol & Pharmacol, Sect Receptor Biol & Signaling, S-17165 Stockholm, Sweden.
    A conserved molecular switch in Class F receptors regulates receptor activation and pathway selection2019In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 10, article id 667Article in journal (Refereed)
    Abstract [en]

    Class F receptors are considered valuable therapeutic targets due to their role in human disease, but structural changes accompanying receptor activation remain unexplored. Employing population and cancer genomics data, structural analyses, molecular dynamics simulations, resonance energy transfer-based approaches and mutagenesis, we identify a conserved basic amino acid in TM6 in Class F receptors that acts as a molecular switch to mediate receptor activation. Across all tested Class F receptors (FZD(4,5,6,7,) SMO), mutation of the molecular switch confers an increased potency of agonists by stabilizing an active conformation as assessed by engineered mini G proteins as conformational sensors. Disruption of the switch abrogates the functional interaction between FZDs and the phosphoprotein Dishevelled, supporting conformational selection as a prerequisite for functional selectivity. Our studies reveal the molecular basis of a common activation mechanism conserved in all Class F receptors, which facilitates assay development and future discovery of Class F receptor-targeting drugs.

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