Structural Aspects of Photopharmacology: Insight into the Binding of Photoswitchable and Photocaged Inhibitors to the Glutamate Transporter HomologueClick to copy article linkArticle link copied!
- Valentina ArkhipovaValentina ArkhipovaUniversity Medical Center Groningen, Department of Radiology, Medical Imaging Center, University of Groningen, Hanzeplein 1, 9713 GZ Groningen, The NetherlandsGroningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The NetherlandsMore by Valentina Arkhipova
- Haigen FuHaigen FuDepartment of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The NetherlandsMore by Haigen Fu
- Mark W. H. HoorensMark W. H. HoorensUniversity Medical Center Groningen, Department of Radiology, Medical Imaging Center, University of Groningen, Hanzeplein 1, 9713 GZ Groningen, The NetherlandsStratingh Institute for Chemistry, Faculty of Science and Engineering, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The NetherlandsMore by Mark W. H. Hoorens
- Gianluca TrincoGianluca TrincoGroningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The NetherlandsMore by Gianluca Trinco
- Lucien N. LameijerLucien N. LameijerUniversity Medical Center Groningen, Department of Radiology, Medical Imaging Center, University of Groningen, Hanzeplein 1, 9713 GZ Groningen, The NetherlandsStratingh Institute for Chemistry, Faculty of Science and Engineering, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The NetherlandsMore by Lucien N. Lameijer
- Egor MarinEgor MarinGroningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The NetherlandsResearch Center for Molecular Mechanisms of Aging and Age-related Diseases, Moscow Institute of Physics and Technology, Dolgoprudny 141701, RussiaMore by Egor Marin
- Ben L. FeringaBen L. FeringaStratingh Institute for Chemistry, Faculty of Science and Engineering, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The NetherlandsMore by Ben L. Feringa
- Gerrit J. PoelarendsGerrit J. PoelarendsDepartment of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The NetherlandsMore by Gerrit J. Poelarends
- Wiktor Szymanski*Wiktor Szymanski*Email [email protected]University Medical Center Groningen, Department of Radiology, Medical Imaging Center, University of Groningen, Hanzeplein 1, 9713 GZ Groningen, The NetherlandsStratingh Institute for Chemistry, Faculty of Science and Engineering, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The NetherlandsMore by Wiktor Szymanski
- Dirk J. Slotboom*Dirk J. Slotboom*Email [email protected]Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The NetherlandsMore by Dirk J. Slotboom
- Albert Guskov*Albert Guskov*Email [email protected]Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The NetherlandsResearch Center for Molecular Mechanisms of Aging and Age-related Diseases, Moscow Institute of Physics and Technology, Dolgoprudny 141701, RussiaMore by Albert Guskov
Abstract
Photopharmacology addresses the challenge of drug selectivity and side effects through creation of photoresponsive molecules activated with light with high spatiotemporal precision. This is achieved through incorporation of molecular photoswitches and photocages into the pharmacophore. However, the structural basis for the light-induced modulation of inhibitory potency in general is still missing, which poses a major design challenge for this emerging field of research. Here we solved crystal structures of the glutamate transporter homologue GltTk in complex with photoresponsive transport inhibitors—azobenzene derivative of TBOA (both in trans and cis configuration) and with the photocaged compound ONB-hydroxyaspartate. The essential role of glutamate transporters in the functioning of the central nervous system renders them potential therapeutic targets in the treatment of neurodegenerative diseases. The obtained structures provide a clear structural insight into the origins of photocontrol in photopharmacology and lay the foundation for application of photocontrolled ligands to study the transporter dynamics by using time-resolved X-ray crystallography.
Introduction
Results
GltTk Crystal Structures in Complex with Photoswitches
GltTk Crystal Structure in Complex with a Photocaged Inhibitor
Discussion
Materials and Methods
Protein Purification and Crystallization
Ligand Docking
Data Collection and Structure Determination
Transport Assay
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.0c11336.
Tables S1 and S2; Figures S1–S4, and information about synthesis (PDF)
The structural models and associated structure factors are available at PDB data bank under the following accession codes: 6ZLH, 6ZL4, and 6ZGB.
Terms & Conditions
Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.
Acknowledgments
H.F. acknowledges funding from the China Scholarship Council; E.M. and A.G. acknowledge support from RFBR (19-29-12022).
GltTk | glutamate transporter homologue from Thermococcus kodakarensis |
GltPh | glutamate transporter homologue from Phyrococcus horikoshii |
SLC | solute carrier |
GPCR | G-protein coupled receptor |
ONB | o-nitrobenzyl |
BODIPY | boron dipyrromethene |
PPG | photoremovable protecting group |
TRPV | transient receptor potential vanilloid channel |
EAAT | excitatory amino acid transporter |
ASCT | alanine-serine-cystein transporter |
TBOA | dl-threo-β-benzyloxyaspartic acid |
TFB-TBOA | (3S)-3-[[3-[[4-(trifluoromethyl)benzoyl]amino]phenyl]methoxy]-l-aspartic acid. |
References
This article references 71 other publications.
- 1Velema, W. A.; Van Der Berg, J. P.; Hansen, M. J.; Szymanski, W.; Driessen, A. J. M.; Feringa, B. L. Optical Control of Antibacterial Activity. Nat. Chem. 2013, 5 (11), 924– 928, DOI: 10.1038/nchem.1750Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsVerurzN&md5=ab3ff514012f788c49981666d78acccbOptical control of antibacterial activityVelema, Willem A.; van der Berg, Jan Pieter; Hansen, Mickel J.; Szymanski, Wiktor; Driessen, Arnold J. M.; Feringa, Ben L.Nature Chemistry (2013), 5 (11), 924-928CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)Bacterial resistance is a major problem in the modern world, stemming in part from the build-up of antibiotics in the environment. Novel mol. approaches that enable an externally triggered increase in antibiotic activity with high spatiotemporal resoln. and auto-inactivation are highly desirable. Here, the authors report a responsive, broad-spectrum, antibacterial agent that can be temporally activated with light, whereupon it auto-inactivates on the scale of hours. The use of such a smart antibiotic might prevent the build-up of active antimicrobial material in the environment. Reversible optical control over active drug concn. enables us to obtain pharmacodynamic information. Precisely localized control of activity is achieved, allowing the growth of bacteria to be confined to defined patterns, which has potential for the development of treatments that avoid interference with the endogenous microbial population in other parts of the organism.
- 2Borowiak, M.; Nahaboo, W.; Reynders, M.; Nekolla, K.; Jalinot, P.; Hasserodt, J.; Rehberg, M.; Delattre, M.; Zahler, S.; Vollmar, A.; Trauner, D.; Thorn-Seshold, O. Photoswitchable Inhibitors of Microtubule Dynamics Optically Control Mitosis and Cell Death. Cell 2015, 162 (2), 403– 411, DOI: 10.1016/j.cell.2015.06.049Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtFCkur7M&md5=0fdc2af4eb68494cbf55e6383a4ecd47Photoswitchable Inhibitors of Microtubule Dynamics Optically Control Mitosis and Cell DeathBorowiak, Malgorzata; Nahaboo, Wallis; Reynders, Martin; Nekolla, Katharina; Jalinot, Pierre; Hasserodt, Jens; Rehberg, Markus; Delattre, Marie; Zahler, Stefan; Vollmar, Angelika; Trauner, Dirk; Thorn-Seshold, OliverCell (Cambridge, MA, United States) (2015), 162 (2), 403-411CODEN: CELLB5; ISSN:0092-8674. (Cell Press)Small mols. that interfere with microtubule dynamics, such as Taxol and the Vinca alkaloids, are widely used in cell biol. research and as clin. anticancer drugs. However, their activity cannot be restricted to specific target cells, which also causes severe side effects in chemotherapy. Here, the authors introduce the photostatins, inhibitors that can be switched on and off in vivo by visible light, to optically control microtubule dynamics. Photostatins modulate microtubule dynamics with a subsecond response time and control mitosis in living organisms with single-cell spatial precision. In longer-term applications in cell culture, photostatins are up to 250 times more cytotoxic when switched on with blue light than when kept in the dark. Therefore, photostatins are both valuable tools for cell biol., and are promising as a new class of precision chemotherapeutics whose toxicity may be spatiotemporally constrained using light.
- 3Hoorens, M. W. H.; Szymanski, W. Reversible, Spatial and Temporal Control over Protein Activity Using Light. Trends Biochem. Sci. 2018, 43 (8), 567– 575, DOI: 10.1016/j.tibs.2018.05.004Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtFSru7vI&md5=6802043365a055b6a166db2f493fb391Reversible, Spatial and Temporal Control over Protein Activity Using LightHoorens, Mark W. H.; Szymanski, WiktorTrends in Biochemical Sciences (2018), 43 (8), 567-575CODEN: TBSCDB; ISSN:0968-0004. (Elsevier Ltd.)A review. In biomedical sciences, the function of a protein of interest is investigated by altering its net activity and assessing the consequences for the cell or organism. To change the activity of a protein, a wide variety of chem. and genetic tools have been developed. The drawback of most of these tools is that they do not allow for reversible, spatial and temporal control. Here, we describe selected developments in photopharmacol. that aim at establishing such control over protein activity through bioactive mols. with photo-controlled potency. We also discuss why such control is desired and what challenges still need to be overcome for photopharmacol. to reach its maturity as a chem. biol. research tool.
- 4Lerch, M. M.; Hansen, M. J.; van Dam, G. M.; Szymanski, W.; Feringa, B. L. Emerging Targets in Photopharmacology. Angew. Chem., Int. Ed. 2016, 55 (37), 10978– 10999, DOI: 10.1002/anie.201601931Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtFSqurnN&md5=f2939978a74c93e46e89ccbf6ac9dde4Emerging Targets in PhotopharmacologyLerch, Michael M.; Hansen, Mickel J.; van Dam, Gooitzen M.; Szymanski, Wiktor; Feringa, Ben L.Angewandte Chemie, International Edition (2016), 55 (37), 10978-10999CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The field of photopharmacol. uses mol. photoswitches to establish control over the action of bioactive mols. It aims to reduce systemic drug toxicity and the emergence of resistance, while achieving unprecedented precision in treatment. By using small mols., photopharmacol. provides a viable alternative to optogenetics. We present here a crit. overview of the different pharmacol. targets in various organs and a survey of organ systems in the human body that can be addressed in a non-invasive manner. We discuss the prospects for the selective delivery of light to these organs and the specific requirements for light-activatable drugs. We also aim to illustrate the druggability of medicinal targets with recent findings and emphasize where conceptually new approaches have to be explored to provide photopharmacol. with future opportunities to bring "smart" mol. design ultimately to the realm of clin. use.
- 5Tochitsky, I.; Kienzler, M. A.; Isacoff, E.; Kramer, R. H. Restoring Vision to the Blind with Chemical Photoswitches. Chem. Rev. 2018, 118 (21), 10748– 10773, DOI: 10.1021/acs.chemrev.7b00723Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtVOhsrfI&md5=a953df81c5a7e02a126aad3b14ca9e4fRestoring vision to the blind with chemical photoswitchesTochitsky, Ivan; Kienzler, Michael A.; Isacoff, Ehud; Kramer, Richard H.Chemical Reviews (Washington, DC, United States) (2018), 118 (21), 10748-10773CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Degenerative retinal diseases such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD) affect millions of people around the world and lead to irreversible vision loss if left untreated. A no. of therapeutic strategies have been developed over the years to treat these diseases or restore vision to already blind patients. In this Review, we describe the development and translational application of light-sensitive chem. photoswitches to restore visual function to the blind retina and compare the translational potential of photoswitches with other vision-restoring therapies. This therapeutic strategy is enabled by an efficient fusion of chem. synthesis, chem. biol., and mol. biol. and is broadly applicable to other biol. systems. We hope this Review will be of interest to chemists as well as neuroscientists and clinicians.
- 6Hüll, K.; Morstein, J.; Trauner, D. In Vivo Photopharmacology. Chem. Rev. 2018, 118 (21), 10710– 10747, DOI: 10.1021/acs.chemrev.8b00037Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXht1OmsrrK&md5=9d82ae8b341d68f2e88ec4f6bf3915e0In Vivo PhotopharmacologyHull, Katharina; Morstein, Johannes; Trauner, DirkChemical Reviews (Washington, DC, United States) (2018), 118 (21), 10710-10747CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Synthetic photoswitches have been known for many years, but their usefulness in biol., pharmacol., and medicine has only recently been systematically explored. Over the past decade photopharmacol. has grown into a vibrant field. As the photophys., pharmacodynamic, and pharmacokinetic properties of photoswitches, such as azobenzenes, have become established, they have been applied to a wide range of biol. targets. These include transmembrane proteins (ion channels, transporters, G protein-coupled receptors, receptor-linked enzymes), sol. proteins (kinases, proteases, factors involved in epigenetic regulation), lipid membranes, and nucleic acids. In this review, the authors provide an overview of photopharmacol. using synthetic switches that have been applied in vivo, i.e., in living cells and organisms. The authors discuss the scope and limitations of this approach to study biol. function and the challenges it faces in translational medicine. The relationships between synthetic photoswitches, natural chromophores used in optogenetics, and caged ligands are addressed.
- 7Lundstrom, K. Structural Genomics and Drug Discovery: Molecular Pharmacology. J. Cell. Mol. Med. 2007, 11 (2), 224– 238, DOI: 10.1111/j.1582-4934.2007.00028.xGoogle Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXmsFykurw%253D&md5=b2f77376a2586099f6f473d183a9403eStructural genomics and drug discoveryLundstrom, K.Journal of Cellular and Molecular Medicine (2007), 11 (2), 224-238CODEN: JCMMC9; ISSN:1582-1838. (Blackwell Publishing Ltd.)A review. Structure detn. has already proven useful for lead optimization and direct drug design. The no. of high-resoln. structures available in public databases today exceeds 30,000 and will definitely aid in structure-based drug design. Structural genomics approaches covering whole genomes, topol. similar proteins or gene families are great assets for further progress in the development of new drugs. However, membrane proteins representing 70% of current drug targets are poorly characterized structurally. The problems have been related to difficulties in obtaining large amt. of recombinant membrane proteins as well as their purifn. and structure detn. Structural genomics has proven successful in developing new methods in areas from expression to structure detn. by studying a large no. of target proteins in parallel.
- 8Gomez-Santacana, X.; de Munnik, S. M; Mocking, T. A M; Hauwert, N. J; Sun, S.; Vijayachandran, P.; de Esch, I. J P; Vischer, H. F; Wijtmans, M.; Leurs, R. A Toolbox of Molecular Photoswitches to Modulate the CXCR3 Chemokine Receptor with Light. Beilstein J. Org. Chem. 2019, 15 (1), 2509– 2523, DOI: 10.3762/bjoc.15.244Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitlagtbnE&md5=36f7e7dcf3dd60ddc10a2c3349a3a79aA toolbox of molecular photoswitches to modulate the CXCR3 chemokine receptor with lightGomez-Santacana, Xavier; de Munnik, Sabrina M.; Mocking, Tamara A. M.; Hauwert, Niels J.; Sun, Shanliang; Vijayachandran, Prashanna; de Esch, Iwan J. P.; Vischer, Henry F.; Wijtmans, Maikel; Leurs, RobBeilstein Journal of Organic Chemistry (2019), 15 (), 2509-2523CODEN: BJOCBH; ISSN:1860-5397. (Beilstein-Institut zur Foerderung der Chemischen Wissenschaften)A detailed structure-activity relationship for the scaffold of VUF16216, previously communicated as a small-mol. efficacy photoswitch for the peptidergic chemokine GPCR CXCR3 was reported. A series of photoswitchable azobenzene ligands was prepd. through various synthetic strategies and multistep syntheses. Photochem. and pharmacol. properties were used to guide the design iterations. Investigations of positional and substituent effects reveal that halogen substituents on the ortho-position of the outer ring are preferred for conferring partial agonism on the cis form of the ligands. This effect could be expanded by an electron-donating group on the para-position of the central ring. A variety of efficacy differences between the trans and cis forms emerges from these compds. Tool compds. VUF15888 and VUF16620 represent more subtle efficacy switches, while VUF16216 displays the largest efficacy switch, from antagonism to full agonism. The compd. class disclosed here can aid in new photopharmacol. studies of CXCR3 signaling.
- 9Ricart-Ortega, M.; Font, J.; Llebaria, A. GPCR Photopharmacology. Mol. Cell. Endocrinol. 2019, 488, 36– 51, DOI: 10.1016/j.mce.2019.03.003Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXltVSgu7g%253D&md5=b11852d80aee99387d1a01a44ab29d61GPCR photopharmacologyRicart-Ortega, Maria; Font, Joan; Llebaria, AmadeuMolecular and Cellular Endocrinology (2019), 488 (), 36-51CODEN: MCEND6; ISSN:0303-7207. (Elsevier Ireland Ltd.)New technologies for spatial and temporal remote control of G protein-coupled receptors (GPCRs) are necessary to unravel the complexity of GPCR signalling in cells, tissues and living organisms. An effective approach, recently developed, consists on the design of light-operated ligands whereby light-dependent GPCR activity regulation can be achieved. In this context, the use of light provides an advantage as it combines safety, easy delivery, high resoln. and it does not interfere with most cellular processes. In this review we summarize the most relevant successful achievements in GPCR photopharmacol. These recent findings constitute a significant advance in research studies on the mol. dynamics of receptor activation and their physiol. roles in vivo. Moreover, these mols. hold potential toward clin. uses as light-operated drugs, which can overcome some of the problems of conventional pharmacol.
- 10Hauwert, N. J.; Mocking, T. A. M.; Da Costa Pereira, D.; Kooistra, A. J.; Wijnen, L. M.; Vreeker, G. C. M.; Verweij, E. W. E.; De Boer, A. H.; Smit, M. J.; De Graaf, C.; Vischer, H. F.; de Esch, I. J. P.; Wijtmans, M.; Leurs, R. Synthesis and Characterization of a Bidirectional Photoswitchable Antagonist Toolbox for Real-Time GPCR Photopharmacology. J. Am. Chem. Soc. 2018, 140 (12), 4232– 4243, DOI: 10.1021/jacs.7b11422Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXjt1ymtLY%253D&md5=901f689b2d81105aa479089988551a80Synthesis and Characterization of a Bidirectional Photoswitchable Antagonist Toolbox for Real-Time GPCR PhotopharmacologyHauwert, Niels J.; Mocking, Tamara A. M.; Da Costa Pereira, Daniel; Kooistra, Albert J.; Wijnen, Lisa M.; Vreeker, Gerda C. M.; Verweij, Eleonore W. E.; De Boer, Albertus H.; Smit, Martine J.; De Graaf, Chris; Vischer, Henry F.; de Esch, Iwan J. P.; Wijtmans, Maikel; Leurs, RobJournal of the American Chemical Society (2018), 140 (12), 4232-4243CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Noninvasive methods to modulate G protein-coupled receptors (GPCRs) with temporal and spatial precision are in great demand. Photopharmacol. uses photons to control in situ the biol. properties of photoswitchable small-mol. ligands which bodes well for chem. biol. precision approaches. Integrating the light-switchable configurational properties of an azobenzene into the ligand core, the authors developed a bi-directional antagonist toolbox for an archetypical family A GPCR, the histamine H3 receptor (H3R). From 16 newly synthesized photoswitchable compds., VUF14738 (28) and VUF14862 (33) were selected as they swiftly and reversibly photoisomerize and show over 10-fold increased or decreased H3R binding affinities, resp., upon illumination at 360 nm. Both ligands combine long thermal half-lives with fast and high photochem. trans-/cis conversion, allowing their use in real-time electrophysiol. expts. with oocytes to confirm dynamic photomodulation of H3R activation in repeated second-scale cycles. VUF14738 and VUF14862 are robust and fatigue-resistant photoswitchable GPCR antagonists suitable for spatio-temporal studies of H3R signaling.
- 11Bregestovski, P. D.; Maleeva, G. V. Photopharmacology: A Brief Review Using the Control of Potassium Channels as an Example. Neurosci. Behav. Physiol. 2019, 49 (2), 184– 191, DOI: 10.1007/s11055-019-00713-3Google ScholarThere is no corresponding record for this reference.
- 12Sansalone, L.; Zhao, J.; Richers, M. T.; Ellis-Davies, G. C. R. Chemical Tuning of Photoswitchable Azobenzenes: A Photopharmacological Case Study Using Nicotinic Transmission. Beilstein J. Org. Chem. 2019, 15 (1), 2812– 2821, DOI: 10.3762/bjoc.15.274Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitlOqsrjK&md5=85243ba927d37afd01dc1a6a19516e91Chemical tuning of photoswitchable azobenzenes: a photopharmacological case study using nicotinic transmissionSansalone, Lorenzo; Zhao, Jun; Richers, Matthew T.; Ellis-Davies, Graham C. R.Beilstein Journal of Organic Chemistry (2019), 15 (), 2812-2821CODEN: BJOCBH; ISSN:1860-5397. (Beilstein-Institut zur Foerderung der Chemischen Wissenschaften)We have developed photochromic probes for the nicotinic acetylcholine receptor that exploit the unique chem. properties of the tetrafluoroazobenzene (4FAB) scaffold. UV light switching and rapid thermal relaxation of the metastable cis configuration are the main drawbacks assocd. with std. AB-based switches. We designed our photoprobes to take advantage of the excellent thermodn. stability of the cis-4FAB configuration (thermal half-life > 12 days at 37 °C in physiol. buffer) and cis-trans photostationary states above 84%. Furthermore, the well-sepd. n-π* absorption bands of trans- and cis-4FAB allow facile photoswitching with visible light in two optical channels. A convergent 11-step synthetic approach allowed the installation of a trimethylammonium (TA) head onto the 4FAB scaffold, by means of an alkyl spacer, to afford a free diffusible 4FABTA probe. TAs are known to agonize nicotinic receptors, so 4FABTA was tested on mouse brain slices and enabled reversible receptor activation with cycles of violet and green light. Due to the very long-lived metastable cis configuration, 4FAB in vivo use could be of great promise for long term biol. studies. Further chem. functionalization of this 4FAB probe with a maleimide functionality allowed clean crosslinking with glutathione. However, attempts to conjugate with a cysteine on a genetically modified nicotinic acetylcholine receptor did not afford the expected light-responsive channel. Our data indicate that the 4FAB photoswitch can be derivatized bifunctionally for genetically-targeted photopharmacol. while preserving all the favorable photophys. properties of the parent 4FAB scaffold, however, the tetrafluoro motif can significantly perturb pharmacophore-protein interactions. In contrast, we found that the freely diffusible 4FABTA probe could be pre-set with green light into an OFF state that was biol. inert, irradn. with violet light effectively "uncaged" agonist activity, but in a photoreversible manner. Since the neurotransmitter acetylcholine has fully satd. heteroatom valences, our photoswitchable 4FABTA probe could be useful for physiol. studies of this neurotransmitter.
- 13Podewin, T.; Broichhagen, J.; Frost, C.; Groneberg, D.; Ast, J.; Meyer-Berg, H.; Fine, N. H. F.; Friebe, A.; Zacharias, M.; Hodson, D. J.; Trauner, D.; Hoffmann-Röder, A. Optical Control of a Receptor-Linked Guanylyl Cyclase Using a Photoswitchable Peptidic Hormone. Chem. Sci. 2017, 8 (6), 4644– 4653, DOI: 10.1039/C6SC05044AGoogle Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmt1ektbo%253D&md5=5f5c7bde048fbd8c90696c275f0f0fbaOptical control of a receptor-linked guanylyl cyclase using a photoswitchable peptidic hormonePodewin, Tom; Broichhagen, Johannes; Frost, Christina; Groneberg, Dieter; Ast, Julia; Meyer-Berg, Helena; Fine, Nicholas H. F.; Friebe, Andreas; Zacharias, Martin; Hodson, David J.; Trauner, Dirk; Hoffmann-Roeder, AnjaChemical Science (2017), 8 (6), 4644-4653CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)The optical control over biol. function with small photoswitchable mols. has gathered significant attention in the last decade. Herein, we describe the design and synthesis of a small library of photoswitchable peptidomimetics based upon human atrial natriuretic peptide (ANP), in which the photochromic amino acid [3-(3-aminomethyl)phenylazo]phenylacetic acid (AMPP) is incorporated into the peptide backbone. The endogeneous hormone ANP signals via the natriuretic peptide receptor A (NPR-A) through raising intracellular cGMP concns., and is involved in blood pressure regulation and sodium homeostasis, as well as lipid metab. and pancreatic function. The cis- and trans-isomers of one of our peptidomimetics, termed TOP271, exhibit a four-fold difference in NPR-A mediated cGMP synthesis in vitro. Despite this seemingly small difference, TOP271 enables large, optically-induced conformational changes ex vivo and transforms the NPR-A into an endogenous photoswitch. Thus, application of TOP271 allows the reversible generation of cGMP using light and remote control can be afforded over vasoactivity in explanted murine aortic rings, as well as pancreatic beta cell function in islets of Langerhans. This study demonstrates the broad applicability of TOP271 to enzyme-dependent signalling processes, extends the toolbox of photoswitchable mols. to all classes of transmembrane receptors and utilizes photopharmacol. to deduce receptor activation on a mol. level.
- 14Beharry, A. A.; Woolley, G. A. Azobenzene Photoswitches for Biomolecules. Chem. Soc. Rev. 2011, 40 (8), 4422– 4437, DOI: 10.1039/c1cs15023eGoogle Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXovVemurg%253D&md5=49407115a7f1bc77edbdba90a79c1e58Azobenzene photoswitches for biomoleculesBeharry, Andrew A.; Woolley, G. AndrewChemical Society Reviews (2011), 40 (8), 4422-4437CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. The photoisomerization of azobenzene has been known for almost 75 years but only recently has this process been widely applied to biol. systems. The central challenge of how to productively couple the isomerization process to a large functional change in a biomol. has been met in a no. of instances and it appears that effective photocontrol of a large variety of biomols. may be possible. This crit. review summarizes key properties of azobenzene that enable its use as a photoswitch in biol. systems and describes strategies for using azobenzene photoswitches to drive functional changes in peptides, proteins, nucleic acids, lipids, and carbohydrates (192 refs.).
- 15Babii, O.; Afonin, S.; Berditsch, M.; Reiβer, S.; Mykhailiuk, P. K.; Kubyshkin, V. S.; Steinbrecher, T.; Ulrich, A. S.; Komarov, I. V. Controlling Biological Activity with Light: Diarylethene-Containing Cyclic Peptidomimetics. Angew. Chem., Int. Ed. 2014, 53 (13), 3392– 3395, DOI: 10.1002/anie.201310019Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXivVertLk%253D&md5=d2e74d71fbe9092e9a531545b4905e28Controlling Biological Activity with Light: Diarylethene-Containing Cyclic PeptidomimeticsBabii, Oleg; Afonin, Sergii; Berditsch, Marina; Reisser, Sabine; Mykhailiuk, Pavel K.; Kubyshkin, Vladimir S.; Steinbrecher, Thomas; Ulrich, Anne S.; Komarov, Igor V.Angewandte Chemie, International Edition (2014), 53 (13), 3392-3395CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Photobiol. processes in nature are usually triggered by nonpeptidic chromophores or by modified side chains. A system is presented in which the polypeptide backbone itself can be conformationally switched by light. An amino acid analog was designed and synthesized based on a reversibly photoisomerizable diarylethene scaffold. This analog was incorporated into the cyclic backbone of the antimicrobial peptide gramicidin S at several sites. The biol. activity of the resulting peptidomimetics could then be effectively controlled by UV/visible light within strictly defined spatial and temporal limits.
- 16Klán, P.; Šolomek, T.; Bochet, C. G.; Blanc, A.; Givens, R.; Rubina, M.; Popik, V.; Kostikov, A.; Wirz, J. Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy. Chem. Rev. 2013, 113 (1), 119– 191, DOI: 10.1021/cr300177kGoogle Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhvVyiurvL&md5=b8defe84c6c629e44d429f82fa4eb5f0Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and EfficacyKlan, Petr; Solomek, Tomas; Bochet, Christian G.; Blanc, Aurelien; Givens, Richard; Rubina, Marina; Popik, Vladimir; Kostikov, Alexey; Wirz, JakobChemical Reviews (Washington, DC, United States) (2013), 113 (1), 119-191CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Photoremovable (sometimes called photoreleasable, photocleavable or photoactivatable) protecting groups (PPGs) provide spatial and temporal control over the release of various chems. such as bioagents (neurotransmitters and cell-signaling mols.), acids, bases, Ca2+ ions, oxidants, insecticides, phermones, fragrances, etc. The present review covers recent developments in the field, focusing on the scope, limitations, and applications of PPGs, which are used to release org. mols. Simplified basic structures of PPGs discussed in this review are provided.
- 17Slanina, T.; Shrestha, P.; Palao, E.; Kand, D.; Peterson, J. A.; Dutton, A. S.; Rubinstein, N.; Weinstain, R.; Winter, A. H.; Klán, P. In Search of the Perfect Photocage: Structure-Reactivity Relationships in Meso-Methyl BODIPY Photoremovable Protecting Groups. J. Am. Chem. Soc. 2017, 139 (42), 15168– 15175, DOI: 10.1021/jacs.7b08532Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhs1Gkur%252FL&md5=93b735b32b8eb44c18391d36fdf0cc47In search of the perfect photocage: structure-reactivity relationships in meso-methyl BODIPY photoremovable protecting groupsSlanina, Tomas; Shrestha, Pradeep; Palao, Eduardo; Kand, Dnyaneshwar; Peterson, Julie A.; Dutton, Andrew S.; Rubinstein, Naama; Weinstain, Roy; Winter, Arthur H.; Klan, PetrJournal of the American Chemical Society (2017), 139 (42), 15168-15175CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)A detailed investigation of the photophys. parameters and photochem. reactivity of meso-Me BODIPY photoremovable protecting groups was accomplished through systematic variation of the leaving group (LG) and core substituents as well as substitutions at boron. Efficiencies of the LG release were evaluated using both steady-state and transient absorption spectroscopies as well as computational analyses to identify the optimal structural features. We find that the quantum yields for photorelease with this photocage are highly sensitive to substituent effects. In particular, we find that the quantum yields of photorelease are improved with derivs. with higher intersystem crossing quantum yields, which can be promoted by core heavy atoms. Moreover, release quantum yields are dramatically improved by boron alkylation, whereas alkylation in the meso-Me position has no effect. Better LGs are released considerably more efficiently than poorer LGs. We find that these substituent effects are additive, for example, a 2,6-diiodo-B-dimethyl BODIPY photocage features quantum yields of 28% for the mediocre LG acetate and a 95% quantum yield of release for chloride. The high chem. and quantum yields combined with the outstanding absorption properties of BODIPY dyes lead to photocages with uncaging cross sections over 10 000 M-1 cm-1, values that surpass cross sections of related photocages absorbing visible light. These new photocages, which absorb strongly near the second harmonic of an Nd:YAG laser (532 nm), hold promise for manipulating and interrogating biol. and material systems with the high spatiotemporal control provided by pulsed laser irradn., while avoiding the phototoxicity problems encountered with many UV-absorbing photocages. More generally, the insights gained from this structure-reactivity relationship may aid in the development of new highly efficient photoreactions.
- 18Deiters, A. Principles and Applications of the Photochemical Control of Cellular Processes. ChemBioChem 2010, 11 (1), 47– 53, DOI: 10.1002/cbic.200900529Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhs1SqtLjM&md5=83e714f7b6f4260287f0a17bcb2cdf06Principles and Applications of the Photochemical Control of Cellular ProcessesDeiters, AlexanderChemBioChem (2010), 11 (1), 47-53CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Biol. processes, particularly gene function, are naturally regulated with high spatiotemporal resoln. in single cells and multicellular organisms. The activity of genes, proteins, and other biol. mols. is precisely controlled in timing and location. This is esp. evident during the complex biol. processes obsd. in the development of an organism. In order to understand and to study these processes and their misregulation in human disease, it is imperative to control them with the same level of spatiotemporal resoln. found in nature. Here, light irradn. represents a unique tool, because it can be easily and precisely controlled in timing, location, and amplitude; thus, light enables the precise activation and deactivation of biol. function.
- 19Schehr, M.; Ianes, C.; Weisner, J.; Heintze, L.; Müller, M. P.; Pichlo, C.; Charl, J.; Brunstein, E.; Ewert, J.; Lehr, M.; Baumann, U.; Rauh, D.; Knippschild, U.; Peifer, C.; Herges, R. 2-Azo-, 2-Diazocine-Thiazols and 2-Azo-Imidazoles as Photoswitchable Kinase Inhibitors: Limitations and Pitfalls of the Photoswitchable Inhibitor Approach. Photochem. Photobiol. Sci. 2019, 18 (6), 1398– 1407, DOI: 10.1039/C9PP00010KGoogle Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXlsleltbg%253D&md5=e30895dd8f7470dd07482c461219509a2-Azo-, 2-diazocine-thiazols and 2-azo-imidazoles as photoswitchable kinase inhibitors: limitations and pitfalls of the photoswitchable inhibitor approachSchehr, Miriam; Ianes, Chiara; Weisner, Joern; Heintze, Linda; Mueller, Matthias P.; Pichlo, Christian; Charl, Julia; Brunstein, Elena; Ewert, Julia; Lehr, Marc; Baumann, Ulrich; Rauh, Daniel; Knippschild, Uwe; Peifer, Christian; Herges, RainerPhotochemical & Photobiological Sciences (2019), 18 (6), 1398-1407CODEN: PPSHCB; ISSN:1474-905X. (Royal Society of Chemistry)In photopharmacol., photoswitchable compds. including azobenzene or other diarylazo moieties exhibit bioactivity against a target protein typically in the slender E-configuration, whereas the rather bulky Z-configuration usually is pharmacol. less potent. Herein we report the design, synthesis and photochem./inhibitory characterization of new photoswitchable kinase inhibitors targeting p38α MAPK and CK1δ. A well characterized inhibitor scaffold was used to attach arylazo- and diazocine moieties. When the isolated isomers, or the photostationary state (PSS) of isomers, were tested in commonly used in vitro kinase assays, however, only small differences in activity were obsd. X-ray analyses of ligand-bound p38α MAPK and CK1δ complexes revealed dynamic conformational adaptations of the protein with respect to both isomers. More importantly, irreversible redn. of the azo group to the corresponding hydrazine was obsd. Independent expts. revealed that reducing agents such as DTT (dithiothreitol) and GSH (glutathione) that are typically used for protein stabilization in biol. assays were responsible. Two further sources of error are the concn. dependence of the E-Z-switching efficiency and artifacts due to incomplete exclusion of light during testing. Our findings may also apply to a no. of previously investigated azobenzene-based photoswitchable inhibitors.
- 20Morgan, H. P.; McNae, I. W.; Nowicki, M. W.; Zhong, W.; Michels, P. A. M.; Auld, D. S.; Fothergill-Gilmore, L. A.; Walkinshaw, M. D. The Trypanocidal Drug Suramin and Other Trypan Blue Mimetics Are Inhibitors of Pyruvate Kinases and Bind to the Adenosine Site. J. Biol. Chem. 2011, 286 (36), 31232– 31240, DOI: 10.1074/jbc.M110.212613Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtFaitLbO&md5=5aec6da4436d8ef23a0048ad1f3e6158The Trypanocidal Drug Suramin and Other Trypan Blue Mimetics Are Inhibitors of Pyruvate Kinases and Bind to the Adenosine SiteMorgan, Hugh P.; McNae, Iain W.; Nowicki, Matthew W.; Zhong, Wenhe; Michels, Paul A. M.; Auld, Douglas S.; Fothergill-Gilmore, Linda A.; Walkinshaw, Malcolm D.Journal of Biological Chemistry (2011), 286 (36), 31232-31240, S31232/1-S31232/12CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Ehrlich's pioneering chemotherapeutic expts. published in 1904 (Ehrlich, P., and Shiga, K. (1904) Berlin Klin. Wochenschrift 20, 329-362) described the efficacy of a series of dye mols. including trypan blue and trypan red to eliminate trypanosome infections in mice. The mol. structures of the dyes provided a starting point for the synthesis of suramin, which was developed and used as a trypanocidal drug in 1916 and is still in clin. use. Despite the biol. importance of these dye-like mols., the mode of action on trypanosomes has remained elusive. Here the authors present crystal structures of suramin and three related dyes in complex with pyruvate kinases from Leishmania mexicana or from Trypanosoma cruzi. The Ph sulfonate groups of all four mols. (suramin, Ponceau S, acid blue 80, and benzothiazole-2,5-disulfonic acid) bind in the position of ADP/ATP at the active sites of the pyruvate kinases (PYKs). The binding positions in the two different trypanosomatid PYKs are nearly identical. The authors show that suramin competitively inhibits PYKs from humans (muscle, tumor, and liver isoenzymes, Ki = 1.1-17 μM), T. cruzi (Ki = 108 μM), and L. mexicana (Ki = 116 μM), all of which have similar active sites. Synergistic effects were obsd. when examg. suramin inhibition in the presence of an allosteric effector mol., whereby IC50 values decreased up to 2-fold for both trypanosomatid and human PYKs. These kinetic and structural analyses provide insight into the promiscuous inhibition obsd. for suramin and into the mode of action of the dye-like mols. used in Ehrlich's original expts.
- 21Dubay, K. H.; Iwan, K.; Osorio-Planes, L.; Geissler, P. L.; Groll, M.; Trauner, D.; Broichhagen, J. A Predictive Approach for the Optical Control of Carbonic Anhydrase II Activity. ACS Chem. Biol. 2018, 13 (3), 793– 800, DOI: 10.1021/acschembio.7b00862Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtlyqtbs%253D&md5=f26058ffabe322ca927e715b10b113fcA Predictive Approach for the Optical Control of Carbonic Anhydrase II ActivityDuBay, Kateri H.; Iwan, Katharina; Osorio-Planes, Laura; Geissler, Phillip L.; Groll, Michael; Trauner, Dirk; Broichhagen, JohannesACS Chemical Biology (2018), 13 (3), 793-800CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Optogenetics and photopharmacol. are powerful approaches to investigating biochem. systems. While the former is based on genetically encoded photoreceptors that utilize abundant chromophores, the latter relies on synthetic photoswitches that are either freely diffusible or covalently attached to specific bioconjugation sites, which are often native or engineered cysteines. The identification of suitable cysteine sites and appropriate linkers for attachment is in general a lengthy and cumbersome process. Herein, we describe an in silico screening approach designed to propose a small no. of optimal combinations. By applying this computational approach to human carbonic anhydrase II (hCAII) and a set of three photochromic tethered ligands (PTLs), the no. of potential site-ligand combinations was narrowed from over 750 down to 6, which we then evaluated exptl. Two of these six combinations resulted in light-responsive human Carbonic Anhydrases (LihCAs), which were characterized with enzymic activity assays, mass spectrometry, and X-ray crystallog. Our study also provides insights into the reactivity of cysteines toward maleimides and the hydrolytic stability of the adducts obtained.
- 22Reiter, A.; Skerra, A.; Trauner, D.; Schiefner, A. A Photoswitchable Neurotransmitter Analogue Bound to Its Receptor. Biochemistry 2013, 52 (50), 8972– 8974, DOI: 10.1021/bi4014402Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvVGktLzN&md5=1d77774d2941f27d09018139e820b812A Photoswitchable Neurotransmitter Analogue Bound to Its ReceptorReiter, Alwin; Skerra, Arne; Trauner, Dirk; Schiefner, AndreBiochemistry (2013), 52 (50), 8972-8974CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Incorporation of the azobenzene deriv. gluazo, a synthetic photochromic ligand, into a kainate receptor allows for the optical control of neuronal activity. The crystal structure of gluazo bound to a dimeric GluK2 ligand-binding domain reveals one monomer in a closed conformation, occupied by gluazo, and the other in an open conformation, with a bound buffer mol. The glutamate group of gluazo interacts like the natural glutamate ligand, while its trans-azobenzene moiety protrudes into a tunnel. This elongated cavity presumably cannot accommodate a cis-azobenzene, which explains the reversible activation of the receptor upon photoisomerization.
- 23Cunha, M. R.; Bhardwaj, R.; Lindinger, S.; Butorac, C.; Romanin, C.; Hediger, M. A.; Reymond, J. L. Photoswitchable Inhibitor of the Calcium Channel TRPV6. ACS Med. Chem. Lett. 2019, 10 (9), 1341– 1345, DOI: 10.1021/acsmedchemlett.9b00298Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhsV2jt7jN&md5=df43f1398f3f6acef072e05b539f249bPhotoswitchable Inhibitor of the Calcium Channel TRPV6Cunha, Micael R.; Bhardwaj, Rajesh; Lindinger, Sonja; Butorac, Carmen; Romanin, Christoph; Hediger, Matthias A.; Reymond, Jean-LouisACS Medicinal Chemistry Letters (2019), 10 (9), 1341-1345CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)Herein the authors report the first photoswitchable inhibitor of Transient Receptor Potential Vanilloid 6 (TRPV6), a selective calcium channel involved in a no. of diseases and in cancer progression. By surveying analogs of a previously reported TRPV6 inhibitor appended with a phenyl-diazo group, the authors identified a compd. switching between a weak TRPV6 inhibitor in its dark, E-diazo stereoisomer (Z/E = 3:97, IC50 >> 10 μM) and a potent inhibitor as the Z-diazo stereoisomer accessible reversibly by UV irradn. at λ = 365 nm (Z/E = 3:1, IC50 = 1.7±0.4 μM), thereby allowing precise spatiotemporal control of inhibition. This new tool compd. should be useful to deepen the understanding of TRPV6.
- 24Vandenberg, R. J.; Ryan, R. M. Mechanisms of Glutamate Transport. Physiol. Rev. 2013, 93 (4), 1621– 1657, DOI: 10.1152/physrev.00007.2013Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvVShtrrP&md5=b573b96bbaf6f03c835c471cf2aaf7b8Mechanisms of glutamate transportVandenberg, Robert J.; Ryan, Renae M.Physiological Reviews (2013), 93 (4), 1621-1657CODEN: PHREA7; ISSN:0031-9333. (American Physiological Society)A review. L-Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system and plays important roles in a wide variety of brain functions, but it is also a key player in the pathogenesis of many neurol. disorders. The control of glutamate concns. is crit. to the normal functioning of the central nervous system, and in this review we discuss how glutamate transporters regulate glutamate concns. to maintain dynamic signaling mechanisms between neurons. In 2004, the crystal structure of a prokaryotic homolog of the mammalian glutamate transporter family of proteins was crystd. and its structure detd. This has paved the way for a better understanding of the structural basis for glutamate transporter function. In this review we provide a broad perspective of this field of research, but focus primarily on the more recent studies with a particular emphasis on how our understanding of the structure of glutamate transporters has generated new insights.
- 25Stergachis, A. B.; Pujol-Giménez, J.; Gyimesi, G.; Fuster, D.; Albano, G.; Troxler, M.; Picker, J.; Rosenberg, P. A.; Bergin, A.; Peters, J.; El Achkar, C. M.; Harini, C.; Manzi, S.; Rotenberg, A.; Hediger, M. A.; Rodan, L. H. Recurrent SLC1A2 Variants Cause Epilepsy via a Dominant Negative Mechanism. Ann. Neurol. 2019, 85 (6), 921– 926, DOI: 10.1002/ana.25477Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXpsFSqtrw%253D&md5=b93ff73eae24f869d28d6df954a90f51Recurrent SLC1A2 variants cause epilepsy via a dominant negative mechanismStergachis, Andrew B.; Pujol-Gimenez, Jonai; Gyimesi, Gergely; Fuster, Daniel; Albano, Giusppe; Troxler, Marina; Picker, Jonathan; Rosenberg, Paul A.; Bergin, Ann; Peters, Jurriaan; El Achkar, Christelle Moufawad; Harini, Chellamani; Manzi, Shannon; Rotenberg, Alexander; Hediger, Matthias A.; Rodan, Lance H.Annals of Neurology (2019), 85 (6), 921-926CODEN: ANNED3; ISSN:0364-5134. (John Wiley & Sons, Inc.)SLC1A2 is a trimeric transporter essential for clearing glutamate from neuronal synapses. Recurrent de novo SLC1A2 missense variants cause a severe, early onset developmental and epileptic encephalopathy via an unclear mechanism. We demonstrate that all 3 variants implicated in this condition localize to the trimerization domain of SLC1A2, and that the Leu85Pro variant acts via a dominant neg. mechanism to reduce, but not eliminate, wild-type SLC1A2 protein localization and function. Finally, we demonstrate that treatment of a 20-mo-old SLC1A2-related epilepsy patient with the SLC1A2-modulating agent ceftriaxone did not result in a significant change in daily spasm count.
- 26Zike, I. D.; Chohan, M. O.; Kopelman, J. M.; Krasnow, E. N.; Flicker, D.; Nautiyal, K. M.; Bubser, M.; Kellendonk, C.; Jones, C. K.; Stanwood, G.; Tanaka, K. F.; Moore, H.; Ahmari, S. E.; Veenstra-VanderWeele, J. OCD Candidate Gene SLC1A1/EAAT3 Impacts Basal Ganglia-Mediated Activity and Stereotypic Behavior. Proc. Natl. Acad. Sci. U. S. A. 2017, 114 (22), 5719– 5724, DOI: 10.1073/pnas.1701736114Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXnslKls7k%253D&md5=3e007fbd35b364481472c7ae284a0b82OCD candidate gene SLC1A1/EAAT3 impacts basal ganglia-mediated activity and stereotypic behaviorZike, Isaac D.; Chohan, Muhammad O.; Kopelman, Jared M.; Krasnow, Emily N.; Flicker, Daniel; Nautiyal, Katherine M.; Bubser, Michael; Kellendonk, Christoph; Jones, Carrie K.; Stanwood, Gregg; Tanaka, Kenji Fransis; Moore, Holly; Ahmari, Susanne E.; Veenstra-VanderWeele, JeremyProceedings of the National Academy of Sciences of the United States of America (2017), 114 (22), 5719-5724CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Obsessive-compulsive disorder (OCD) is a chronic, disabling condition with inadequate treatment options that leave most patients with substantial residual symptoms. Structural, neurochem., and behavioral findings point to a significant role for basal ganglia circuits and for the glutamate system in OCD. Genetic linkage and assocn. studies in OCD point to SLC1A1, which encodes the neuronal glutamate/aspartate/cysteine transporter excitatory amino acid transporter 3 (EAAT3)/excitatory amino acid transporter 1 (EAAC1). However, no previous studies have investigated EAAT3 in basal ganglia circuits or in relation to OCD-related behavior. Here, we report a model of Slc1a1 loss based on an excisable STOP cassette that yields successful ablation of EAAT3 expression and function. Using amphetamine as a probe, we found that EAAT3 loss prevents expected increases in (i) locomotor activity, (ii) stereotypy, and (iii) immediate early gene induction in the dorsal striatum following amphetamine administration. Further, Slc1a1-STOP mice showed diminished grooming in an SKF-38393 challenge expt., a pharmacol. model of OCD-like grooming behavior. This reduced grooming is accompanied by reduced dopamine D1 receptor binding in the dorsal striatum of Slc1a1-STOP mice. Slc1a1-STOP mice also exhibit reduced extracellular dopamine concns. in the dorsal striatum both at baseline and following amphetamine challenge. Viral-mediated restoration of Slc1a1/EAAT3 expression in the midbrain but not in the striatum results in partial rescue of amphetamine-induced locomotion and stereotypy in Slc1a1-STOP mice, consistent with an impact of EAAT3 loss on presynaptic dopaminergic function. Collectively, these findings indicate that the most consistently assocd. OCD candidate gene impacts basal ganglia-dependent repetitive behaviors.
- 27Choi, K. D.; Jen, J. C.; Choi, S. Y.; Shin, J. H.; Kim, H. S.; Kim, H. J.; Kim, J. S.; Choi, J. H. Late-Onset Episodic Ataxia Associated with SLC1A3Mutation. J. Hum. Genet. 2017, 62 (3), 443– 446, DOI: 10.1038/jhg.2016.137Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXjsVSlsL4%253D&md5=094d25d768839e8262bb7ab920f78e71Late-onset episodic ataxia associated with SLC1A3 mutationChoi, Kwang-Dong; Jen, Joanna C.; Choi, Seo Young; Shin, Jin-Hong; Kim, Hyang-Sook; Kim, Hyo-Jung; Kim, Ji-Soo; Choi, Jae-HwanJournal of Human Genetics (2017), 62 (3), 443-446CODEN: JHGEFR; ISSN:1434-5161. (Nature Publishing Group)Episodic ataxia type 6 (EA6) is caused by mutations in SLC1A3 that encodes excitatory amino acid transporter 1 (EAAT1), a glial glutamate transporter. EAAT1 regulates the extent and durations of glutamate-mediated signal by the clearance of glutamate after synaptic release. In addn., EAAT1 also has an anion channel activity that prevents addnl. glutamate release. We identified a missense mutation in SLC1A3 in a family with EA. The proband exhibited typical EA2-like symptoms such as recurrent ataxia, slurred speech with a duration of several hours, interictal nystagmus and response to acetazolamide, but had late-onset age of sixth decade. Whole-exome sequencing detected a heterozygous c.1177G>A mutation in SLC1A3. This mutation predicted a substitution of isoleucine for a highly conserved valine residue in the seventh transmembrane domain of EAAT1. The mutation was not present in 100 controls, a large panel of inhouse genome data and various mutation databases. Most functional prediction scores revealed to be deleterious. Same heterozygous mutation was identified in one clin. affected family member and two asymptomatic members. Our data expand the mutation spectrum of SLC1A3 and the clin. phenotype of EA6.
- 28Heimer, G.; Marek-Yagel, D.; Eyal, E.; Barel, O.; Oz Levi, D.; Hoffmann, C.; Ruzzo, E. K.; Ganelin-Cohen, E.; Lancet, D.; Pras, E.; Rechavi, G.; Nissenkorn, A.; Anikster, Y.; Goldstein, D. B.; Ben Zeev, B. SLC1A4Mutations Cause a Novel Disorder of Intellectual Disability, Progressive Microcephaly, Spasticity and Thin Corpus Callosum. Clin. Genet. 2015, 88 (4), 327– 335, DOI: 10.1111/cge.12637Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsFGitLbE&md5=6bca222b398c008b206e1898cd397206SLC1A4 mutations cause a novel disorder of intellectual disability, progressive microcephaly, spasticity and thin corpus callosumHeimer, G.; Marek-Yagel, D.; Eyal, E.; Barel, O.; Oz Levi, D.; Hoffmann, C.; Ruzzo, E. K.; Ganelin-Cohen, E.; Lancet, D.; Pras, E.; Rechavi, G.; Nissenkorn, A.; Anikster, Y.; Goldstein, D. B.; Ben Zeev, B.Clinical Genetics (2015), 88 (4), 327-335CODEN: CLGNAY; ISSN:0009-9163. (Wiley-Blackwell)Two unrelated patients, presenting with significant global developmental delay, severe progressive microcephaly, seizures, spasticity and thin corpus callosum (CC) underwent trio whole-exome sequencing. No candidate variant was found in any known genes related to the phenotype. However, crossing the data of the patients illustrated that they both manifested pathogenic variants in the SLC1A4 gene which codes the ASCT1 transporter of serine and other neutral amino acids. The Ashkenazi patient is homozygous for a deleterious missense c.766G>A, p.(E256K) mutation whereas the Ashkenazi-Iraqi patient is compd. heterozygous for this mutation and a nonsense c.945delTT, p.(Leu315Hisfs*42) mutation. Structural prediction demonstrates truncation of significant portion of the protein by the nonsense mutation and speculates functional disruption by the missense mutation. Both mutations are extremely rare in general population databases, however, the missense mutation was found in heterozygous mode in 1:100 Jewish Ashkenazi controls suggesting a higher carrier rate among Ashkenazi Jews. We conclude that SLC1A4 is the disease causing gene of a novel neurol. disorder manifesting with significant intellectual disability, severe postnatal microcephaly, spasticity and thin CC. The role of SLC1A4 in the serine transport from astrocytes to neurons suggests a possible pathomechanism for this disease and implies a potential therapeutic approach.
- 29Van Geldermalsen, M.; Wang, Q.; Nagarajah, R.; Marshall, A. D.; Thoeng, A.; Gao, D.; Ritchie, W.; Feng, Y.; Bailey, C. G.; Deng, N.; Harvey, K.; Beith, J. M.; Selinger, C. I.; O’Toole, S. A.; Rasko, J. E. J.; Holst, J. ASCT2/SLC1A5 Controls Glutamine Uptake and Tumour Growth in Triple-Negative Basal-like Breast Cancer. Oncogene 2016, 35 (24), 3201– 3208, DOI: 10.1038/onc.2015.381Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhs1eks73J&md5=9f79dc0c47998d477eb1debe4ad3303aASCT2/SLC1A5 controls glutamine uptake and tumor growth in triple-negative basal-like breast cancervan Geldermalsen, M.; Wang, Q.; Nagarajah, R.; Marshall, A. D.; Thoeng, A.; Gao, D.; Ritchie, W.; Feng, Y.; Bailey, C. G.; Deng, N.; Harvey, K.; Beith, J. M.; Selinger, C. I.; O'Toole, S. A.; Rasko, J. E. J.; Holst, J.Oncogene (2016), 35 (24), 3201-3208CODEN: ONCNES; ISSN:0950-9232. (Nature Publishing Group)Alanine, serine, cysteine-preferring transporter 2 (ASCT2; SLC1A5) mediates uptake of glutamine, a conditionally essential amino acid in rapidly proliferating tumor cells. Uptake of glutamine and subsequent glutaminolysis is crit. for activation of the mTORC1 nutrient-sensing pathway, which regulates cell growth and protein translation in cancer cells. This is of particular interest in breast cancer, as glutamine dependence is increased in high-risk breast cancer subtypes. Pharmacol. inhibitors of ASCT2-mediated transport significantly reduced glutamine uptake in human breast cancer cell lines, leading to the suppression of mTORC1 signaling, cell growth and cell cycle progression. Notably, these effects were subtype-dependent, with ASCT2 transport crit. only for triple-neg. (TN) basal-like breast cancer cell growth compared with minimal effects in luminal breast cancer cells. Both stable and inducible shRNA-mediated ASCT2 knockdown confirmed that inhibiting ASCT2 function was sufficient to prevent cellular proliferation and induce rapid cell death in TN basal-like breast cancer cells, but not in luminal cells. Using a bioluminescent orthotopic xenograft mouse model, ASCT2 expression was then shown to be necessary for both successful engraftment and growth of HCC1806 TN breast cancer cells in vivo. Lower tumoral expression of ASCT2 conferred a significant survival advantage in xenografted mice. These responses remained intact in primary breast cancers, where gene expression anal. showed high expression of ASCT2 and glutamine metab.-related genes, including GLUL and GLS, in a cohort of 90 TN breast cancer patients, as well as correlations with the transcriptional regulators, MYC and ATF4. This study provides preclin. evidence for the feasibility of novel therapies exploiting ASCT2 transporter activity in breast cancer, particularly in the high-risk basal-like subgroup of TN breast cancer where there is not only high expression of ASCT2, but also a marked reliance on its activity for sustained cellular proliferation.
- 30Gebhardt, C.; Körner, R.; Heinemann, U. Delayed Anoxic Depolarizations in Hippocampal Neurons of Mice Lacking the Excitatory Amino Acid Carrier 1. J. Cereb. Blood Flow Metab. 2002, 22 (5), 569– 575, DOI: 10.1097/00004647-200205000-00008Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XktlWgsL8%253D&md5=9d98ccc74790c2caf4569be146f84c61Delayed anoxic depolarizations in hippocampal neurons of mice lacking the excitatory amino acid carrier 1Gebhardt, Christine; Korner, Rafael; Heinemann, UweJournal of Cerebral Blood Flow and Metabolism (2002), 22 (5), 569-575CODEN: JCBMDN; ISSN:0271-678X. (Lippincott Williams & Wilkins)Hypoxia leads to a rapid increase in vesicular release of glutamate. In addn., hypoxic glutamate release might be caused by reversed operation of neuronal glutamate transporters. An increase in extracellular glutamate concn. might be an important factor in generating anoxic depolarizations (AD) and subsequent neuronal damage. To study the AD and the vesicular release in hippocampal slices from CD1 wild-type mice and mice in which the neuronal glutamate transporter excitatory amino acid carrier 1 (EAAC1) had been knocked out, the authors performed recordings of field potentials and patch clamp recordings of CA1 pyramidal cells. Latency to anoxic depolarizations was enhanced in EAAC1-/- mice, whereas the hypoxia-induced increase in miniature excitatory postsynaptic current frequency occurred with similarly short latencies and to a similar extent in control and mutated animals. Addnl. block of glial glutamate uptake with TBOA (DL-threo-β-benzyloxyaspartate), a nontransportable and potent inhibitor, dramatically reduced the latency to onset of AD and abolished the difference between wild-type mice and EAAC1-/- mice. The authors conclude that the neuronal glutamate transporter greatly influences the latency to generation of AD. Because ADs are not prevented in EAAC1-deficient mice, vesicular release mechanisms also seem to be involved. They become prominent when glial glutamate transport is blocked.
- 31Bjørn-Yoshimoto, W.; Underhill, S. M. The Importance of the Excitatory Amino Acid Transporter 3 (EAAT3). Neurochem. Int. 2016, 98, 4– 18, DOI: 10.1016/j.neuint.2016.05.007Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2s%252Fkt1KisA%253D%253D&md5=27ff5a73f8de4c0550ebe7ee83660fbdThe importance of the excitatory amino acid transporter 3 (EAAT3)Bjorn-Yoshimoto Walden E; Underhill Suzanne MNeurochemistry international (2016), 98 (), 4-18 ISSN:.The neuronal excitatory amino acid transporter 3 (EAAT3) is fairly ubiquitously expressed in the brain, though it does not necessarily maintain the same function everywhere. It is important in maintaining low local concentrations of glutamate, where its predominant post-synaptic localization can buffer nearby glutamate receptors and modulate excitatory neurotransmission and synaptic plasticity. It is also the main neuronal cysteine uptake system acting as the rate-limiting factor for the synthesis of glutathione, a potent antioxidant, in EAAT3 expressing neurons, while on GABAergic neurons, it is important in supplying glutamate as a precursor for GABA synthesis. Several diseases implicate EAAT3, and modulation of this transporter could prove a useful therapeutic approach. Regulation of EAAT3 could be targeted at several points for functional modulation, including the level of transcription, trafficking and direct pharmacological modulation, and indeed, compounds and experimental treatments have been identified that regulate EAAT3 function at different stages, which together with observations of EAAT3 regulation in patients is giving us insight into the endogenous function of this transporter, as well as the consequences of altered function. This review summarizes work done on elucidating the role and regulation of EAAT3.
- 32Rossi, D. J.; Oshima, T.; Attwell, D. Glutamate Release in Severe Brain Ischaemia Is Mainly by Reversed Uptake. Nature 2000, 403 (6767), 316– 321, DOI: 10.1038/35002090Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD3c7itlantQ%253D%253D&md5=e8d5d2e38a268b4f3540ea8b69b4fa7cGlutamate release in severe brain ischaemia is mainly by reversed uptakeRossi D J; Oshima T; Attwell DNature (2000), 403 (6767), 316-21 ISSN:0028-0836.The release of glutamate during brain anoxia or ischaemia triggers the death of neurons, causing mental or physical handicap. The mechanism of glutamate release is controversial, however. Four release mechanisms have been postulated: vesicular release dependent on external calcium or Ca2+ released from intracellular stores; release through swelling-activated anion channels; an indomethacin-sensitive process in astrocytes; and reversed operation of glutamate transporters. Here we have mimicked severe ischaemia in hippocampal slices and monitored glutamate release as a receptor-gated current in the CA1 pyramidal cells that are killed preferentially in ischaemic hippocampus. Using blockers of the different release mechanisms, we demonstrate that glutamate release is largely by reversed operation of neuronal glutamate transporters, and that it plays a key role in generating the anoxic depolarization that abolishes information processing in the central nervous system a few minutes after the start of ischaemia. A mathematical model incorporating K+ channels, reversible uptake carriers and NMDA (N-methyl-D-aspartate) receptor channels reproduces the main features of the response to ischaemia. Thus, transporter-mediated glutamate homeostasis fails dramatically in ischaemia: instead of removing extracellular glutamate to protect neurons, transporters release glutamate, triggering neuronal death.
- 33Yaster, M.; Guan, X.; Petralia, R. S.; Rothstein, J. D.; Lu, W.; Tao, Y. X. Effect of Inhibition of Spinal Cord Glutamate Transporters on Inflammatory Pain Induced by Formalin and Complete Freund’s Adjuvant. Anesthesiology 2011, 114 (2), 412– 423, DOI: 10.1097/ALN.0b013e318205df50Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtFCisLg%253D&md5=61f279c6e65d9ae4d7eebda860bd831eEffect of inhibition of spinal cord glutamate transporters on inflammatory pain induced by formalin and complete freund's adjuvantYaster, Myron; Guan, Xiaowei; Petralia, Ronald S.; Rothstein, Jeffery D.; Lu, Wei; Tao, Yuan-XiangAnesthesiology (2011), 114 (2), 412-423CODEN: ANESAV; ISSN:0003-3022. (Lippincott Williams & Wilkins)Background: Spinal cord glutamate transporters clear synaptically released glutamate and maintain normal sensory transmission. However, their ultrastructural localization is unknown. Moreover, whether and how they participate in inflammatory pain has not been carefully studied. Methods: Immunogold labeling with electron microscopy was carried out to characterize synaptic and nonsynaptic localization of glutamate transporters in the superficial dorsal horn. Their expression and uptake activity after formalin- and complete Freund's adjuvant (CFA)-induced inflammation were evaluated by Western blot anal. and glutamate uptake assay. Effects of intrathecal glutamate transporter activator (R)-(-)-5-methyl-1-nicotinoyl-2-pyrazoline and inhibitors (DL-threo-β-benzyloxyaspartate [TBOA], dihydrokainate, and DL-threo-β-hydroxyaspartate), or TBOA plus group III metabotropic glutamate receptor antagonist (RS)-α-methylserine-O-phosphate, on formalin- and CFA-induced inflammatory pain were examd. Results: In the superficial dorsal horn, excitatory amino acid carrier 1 is localized in presynaptic membrane, postsynaptic membrane, and axonal and dendritic membranes at nonsynaptic sites, whereas glutamate transporter-1 and glutamate/aspartate transporter are prominent in glial membranes. Although expression of these three spinal glutamate transporters was not altered 1 h after formalin injection or 6 h after CFA injection, glutamate uptake activity was decreased at these time points. Intrathecal (R)-(-)-5-methyl-1-nicotinoyl-2-pyrazoline had no effect on formalin-induced pain behaviors. In contrast, intrathecal TBOA, dihydrokainate, and DL-threo-β-hydroxyaspartate reduced formalin-evoked pain behaviors in the second phase. Intrathecal TBOA also attenuated CFA-induced thermal hyperalgesia at 6 h after CFA injection. The antinociceptive effects of TBOA were blocked by coadministration of (RS)-α-methylserine-O-phosphate. Conclusion: Our findings suggest that spinal glutamate transporter inhibition relieves inflammatory pain through activation of inhibitory presynaptic group III metabotropic glutamate receptors.
- 34Winter, N.; Kovermann, P.; Fahlke, C. A Point Mutation Associated with Episodic Ataxia 6 Increases Glutamate Transporter Anion Currents. Brain 2012, 135 (11), 3416– 3425, DOI: 10.1093/brain/aws255Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3s%252FptVamtg%253D%253D&md5=830b3ac27f7171888bd615534c5b7438A point mutation associated with episodic ataxia 6 increases glutamate transporter anion currentsWinter Natalie; Kovermann Peter; Fahlke ChristophBrain : a journal of neurology (2012), 135 (Pt 11), 3416-25 ISSN:.Episodic ataxia is a human genetic disease characterized by paroxysmal cerebellar incoordination. There are several genetically and clinically distinct forms of this disease, and one of them, episodic ataxia type 6, is caused by mutations in the gene encoding a glial glutamate transporter, the excitatory amino acid transporter-1. So far, reduced glutamate uptake by mutant excitatory amino acid transporter-1 has been thought to be the main pathophysiological process in episodic ataxia type 6. However, excitatory amino acid transporter-1 does not only mediate secondary-active glutamate transport, but also functions as an ion channel. Here, we examined the effects of a disease-associated point mutation, P290R, on glutamate transport, anion current as well as on the subcellular distribution of excitatory amino acid transporter-1 using heterologous expression in mammalian cells. P290R reduces the number of excitatory amino acid transporter-1 in the surface membrane and impairs excitatory amino acid transporter-1-mediated glutamate uptake. Cells expressing P290R excitatory amino acid transporter-1 exhibit larger anion currents than wild-type cells in the absence as well as in the presence of external l-glutamate, despite a lower number of mutant transporters in the surface membrane. Noise analysis revealed unaltered unitary current amplitudes, indicating that P290R modifies opening and closing, and not anion permeation through mutant excitatory amino acid transporter-1 anion channels. These findings identify gain-of-function of excitatory amino acid transporter anion conduction as a pathological process in episodic ataxia. Episodic ataxia type 6 represents the first human disease found to be associated with altered function of excitatory amino acid transporter anion channels and illustrates possible physiological and pathophysiological impacts of this functional mode of this class of glutamate transporters.
- 35Yernool, D.; Boudker, O.; Jin, Y.; Gouaux, E. Structure of a Glutamate Transporter Homologue from Pyrococcus Horikoshii. Nature 2004, 431 (7010), 811– 818, DOI: 10.1038/nature03018Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXotl2ktL4%253D&md5=1818635a8d3cae0ad8c540a912234cb9Structure of a glutamate transporter homologue from Pyrococcus horikoshiiYernool, Dinesh; Boudker, Olga; Jin, Yan; Gouaux, EricNature (London, United Kingdom) (2004), 431 (7010), 811-818CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Glutamate transporters are integral membrane proteins that catalyze the concentrative uptake of glutamate from the synapse to intracellular spaces by harnessing pre-existing ion gradients. In the central nervous system glutamate transporters are essential for normal development and function, and are implicated in stroke, epilepsy and neurodegenerative diseases. Here we present the crystal structure of a eukaryotic glutamate transporter homolog from Pyrococcus horikoshii. The transporter is a bowl-shaped trimer with a solvent-filled extracellular basin extending halfway across the membrane bilayer. At the bottom of the basin are three independent binding sites, each cradled by two helical hairpins, reaching from opposite sides of the membrane. We propose that transport of glutamate is achieved by movements of the hairpins that allow alternating access to either side of the membrane.
- 36Boudker, O.; Ryan, R. M.; Yernool, D.; Shimamoto, K.; Gouaux, E. Coupling Substrate and Ion Binding to Extracellular Gate of a Sodium-Dependent Aspartate Transporter. Nature 2007, 445 (7126), 387– 393, DOI: 10.1038/nature05455Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXos12ntQ%253D%253D&md5=f6ad5a2c7b561a8f38bd907db63640d2Coupling substrate and ion binding to extracellular gate of a sodium-dependent aspartate transporterBoudker, Olga; Ryan, Renae M.; Yernool, Dinesh; Shimamoto, Keiko; Gouaux, EricNature (London, United Kingdom) (2007), 445 (7126), 387-393CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Secondary transporters are integral membrane proteins that catalyze the movement of substrate mols. across the lipid bilayer by coupling substrate transport to one or more ion gradients, thereby providing a mechanism for the concentrative uptake of substrates. Here we describe crystallog. and thermodn. studies of GltPh, a sodium (Na+)-coupled aspartate transporter, defining sites for aspartate, two sodium ions and D,L-threo-β-benzyloxyaspartate, an inhibitor. We further show that helical hairpin 2 is the extracellular gate that controls access of substrate and ions to the internal binding sites. At least two sodium ions bind in close proximity to the substrate and these sodium-binding sites, together with the sodium-binding sites in another sodium-coupled transporter, LeuT, define an unwound α-helix as the central element of the ion-binding motif, a motif well suited to the binding of sodium and to participation in conformational changes that accompany ion binding and unbinding during the transport cycle.
- 37Reyes, N.; Ginter, C.; Boudker, O. Transport Mechanism of a Bacterial Homologue of Glutamate Transporters. Nature 2009, 462 (7275), 880– 885, DOI: 10.1038/nature08616Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhsVentLnE&md5=d663e75e97e7dc7ef66d44b9eb255ea2Transport mechanism of a bacterial homologue of glutamate transportersReyes, Nicolas; Ginter, Christopher; Boudker, OlgaNature (London, United Kingdom) (2009), 462 (7275), 880-885CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Glutamate transporters are integral membrane proteins that catalyze a thermodynamically uphill uptake of the neurotransmitter glutamate from the synaptic cleft into the cytoplasm of glia and neuronal cells by harnessing the energy of pre-existing electrochem. gradients of ions. Crucial to the reaction is the conformational transition of the transporters between outward- and inward-facing states, in which the substrate binding sites are accessible from the extracellular space and the cytoplasm, resp. Here we describe the crystal structure of a double cysteine mutant of a glutamate transporter homolog from Pyrococcus horikoshii, GltPh, which is trapped in the inward-facing state by cysteine crosslinking. Together with the previously detd. crystal structures of GltPh in the outward-facing state, the structure of the crosslinked mutant allows us to propose a mol. mechanism by which GltPh and by analogy, mammalian glutamate transporters, mediate sodium-coupled substrate uptake.
- 38Jensen, S.; Guskov, A.; Rempel, S.; Hänelt, I.; Slotboom, D. J. Crystal Structure of a Substrate-Free Aspartate Transporter. Nat. Struct. Mol. Biol. 2013, 20 (10), 1224– 1226, DOI: 10.1038/nsmb.2663Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtl2ksb%252FN&md5=120e4aa1e318ace8ea037fd946bc6b12Crystal structure of a substrate-free aspartate transporterJensen, Sonja; Guskov, Albert; Rempel, Stephan; Haenelt, Inga; Slotboom, Dirk JanNature Structural & Molecular Biology (2013), 20 (10), 1224-1226CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)Archaeal glutamate transporter homologs catalyze the coupled uptake of aspartate and three sodium ions. After the delivery of the substrate and sodium ions to the cytoplasm, the empty binding site must reorient to the outward-facing conformation to reset the transporter. Here, we report a crystal structure of the substrate-free transporter GltTk from Thermococcus kodakarensis, which provides insight into the mechanism of this essential step in the translocation cycle.
- 39Guskov, A.; Jensen, S.; Faustino, I.; Marrink, S. J.; Slotboom, D. J. Coupled Binding Mechanism of Three Sodium Ions and Aspartate in the Glutamate Transporter Homologue GltTk. Nat. Commun. 2016, 7, 13420, DOI: 10.1038/ncomms13420Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvVGjtL%252FI&md5=98d108c79c739651594f8d55a68cf304Coupled binding mechanism of three sodium ions and aspartate in the glutamate transporter homologue GltTkGuskov, Albert; Jensen, Sonja; Faustino, Ignacio; Marrink, Siewert J.; Slotboom, Dirk JanNature Communications (2016), 7 (), 13420CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)Glutamate transporters catalyze the thermodynamically unfavorable transport of anionic amino acids across the cell membrane by coupling it to the downhill transport of cations. This coupling mechanism is still poorly understood, in part because the available crystal structures of these transporters are of relatively low resoln. Here we solve crystal structures of the archaeal transporter GltTk in the presence and absence of aspartate and use mol. dynamics simulations and binding assays to show how strict coupling between the binding of three sodium ions and aspartate takes place.
- 40Arkhipova, V.; Guskov, A.; Slotboom, D. J. Structural Ensemble of a Glutamate Transporter Homologue in Lipid Nanodisc Environment. Nat. Commun. 2020, 11, 998, DOI: 10.1038/s41467-020-14834-8Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXkvFahtbY%253D&md5=6b3db8911f70dc8416e76274f2f69872Structural ensemble of a glutamate transporter homologue in lipid nanodisc environmentArkhipova, Valentina; Guskov, Albert; Slotboom, Dirk J.Nature Communications (2020), 11 (1), 998CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Abstr.: Glutamate transporters are cation-coupled secondary active membrane transporters that clear the neurotransmitter L-glutamate from the synaptic cleft. These transporters are homotrimers, with each protomer functioning independently by an elevator-type mechanism, in which a mobile transport domain alternates between inward- and outward-oriented states. Using single-particle cryo-EM we have detd. five structures of the glutamate transporter homolog GltTk, a Na+- L-aspartate symporter, embedded in lipid nanodiscs. Dependent on the substrate concns. used, the protomers of the trimer adopt a variety of asym. conformations, consistent with the independent movement. Six of the 15 resolved protomers are in a hitherto elusive state of the transport cycle in which the inward-facing transporters are loaded with Na+ ions. These structures explain how substrate-leakage is prevented - a strict requirement for coupled transport. The belt protein of the lipid nanodiscs bends around the inward oriented protomers, suggesting that membrane deformations occur during transport.
- 41Alleva, C.; Kovalev, K.; Astashkin, R.; Berndt, M. I.; Baeken, C.; Balandin, T.; Gordeliy, V.; Fahlke, C.; Machtens, J. P. Na+-Dependent Gate Dynamics and Electrostatic Attraction Ensure Substrate Coupling in Glutamate Transporters. Sci. Adv. 2020, 6 (47), eaba9854, DOI: 10.1126/sciadv.aba9854Google ScholarThere is no corresponding record for this reference.
- 42Hall, J. L.; Sohail, A.; Cabrita, E. J.; Macdonald, C.; Stockner, T.; Sitte, H. H.; Angulo, J.; MacMillan, F. Saturation Transfer Difference NMR on the Integral Trimeric Membrane Transport Protein GltPh Determines Cooperative Substrate Binding. Sci. Rep. 2020, 10 (1), 1– 9, DOI: 10.1038/s41598-020-73443-zGoogle ScholarThere is no corresponding record for this reference.
- 43Ruan, Y.; Miyagi, A.; Wang, X.; Chami, M.; Boudker, O.; Scheuring, S. Direct Visualization of Glutamate Transporter Elevator Mechanism by High-Speed AFM. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 1584– 1588, DOI: 10.1073/pnas.1616413114Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslejs74%253D&md5=7b9107bb7a775e962fee210070082a76Direct visualization of glutamate transporter elevator mechanism by high-speed AFMRuan, Yi; Miyagi, Atsushi; Wang, Xiaoyu; Chami, Mohamed; Boudker, Olga; Scheuring, SimonProceedings of the National Academy of Sciences of the United States of America (2017), 114 (7), 1584-1588CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Glutamate transporters are essential for recovery of the neurotransmitter glutamate from the synaptic cleft. Crystal structures in the outward- and inward-facing conformations of a glutamate transporter homolog from archaebacterium Pyrococcus horikoshii, sodium/aspartate symporter GltPh, suggested the mol. basis of the transporter cycle. However, dynamic studies of the transport mechanism have been sparse and indirect. Here, the authors present high-speed at. force microscopy (HS-AFM) observations of membrane-reconstituted GltPh at work. HS-AFM movies provided unprecedented real-space and real-time visualization of the transport dynamics. These results showed transport mediated by large amplitude 1.85-nm "elevator" movements of the transport domains consistent with previous crystallog. and spectroscopic studies. Elevator dynamics occur in the absence and presence of Na+ ions and aspartate, but stall in Na+ alone, providing a direct visualization of the ion and substrate symport mechanism. The authors show unambiguously that individual protomers within the trimeric transporter function fully independently.
- 44Garaeva, A. A.; Slotboom, D. J. Elevator-Type Mechanisms of Membrane Transport. Biochem. Soc. Trans. 2020, 48 (3), 1227– 1241, DOI: 10.1042/BST20200290Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsV2ku77M&md5=bf1e3c5e303c1ded7450676e30ac1096Elevator-type mechanisms of membrane transportGaraeva, Alisa A.; Slotboom, Dirk J.Biochemical Society Transactions (2020), 48 (3), 1227-1241CODEN: BCSTB5; ISSN:0300-5127. (Portland Press Ltd.)A review. Membrane transporters are integral membrane proteins that mediate the passage of solutes across lipid bilayers. These proteins undergo conformational transitions between outward- and inward-facing states, which lead to alternating access of the substrate-binding site to the aq. environment on either side of the membrane. Dozens of different transporter families have evolved, providing a wide variety of structural solns. to achieve alternating access. A sub-set of structurally diverse transporters operate by mechanisms that are collectively named 'elevator-type'. These transporters have one common characteristic: they contain a distinct protein domain that slides across the membrane as a rigid body, and in doing so it 'drags' the transported substrate along. Anal. of the global conformational changes that take place in membrane transporters using elevator-type mechanisms reveals that elevator-type movements can be achieved in more than one way. Mol. dynamics simulations and exptl. data help to understand how lipid bilayer properties may affect elevator movements and vice versa.
- 45Esslinger, C. S.; Agarwal, S.; Gerdes, J.; Wilson, P. A.; Davis, E. S.; Awes, A. N.; O’Brien, E.; Mavencamp, T.; Koch, H. P.; Poulsen, D. J.; Rhoderick, J. F.; Chamberlin, A. R.; Kavanaugh, M. P.; Bridges, R. J. The Substituted Aspartate Analogue L-β-Threo-Benzyl-Aspartate Preferentially Inhibits the Neuronal Excitatory Amino Acid Transporter EAAT3. Neuropharmacology 2005, 49 (6), 850– 861, DOI: 10.1016/j.neuropharm.2005.08.009Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtFehsL7I&md5=5bf2793da482cf7bbf27e6a3b0d1bce8The substituted aspartate analogue L-β-threo-benzyl-aspartate preferentially inhibits the neuronal excitatory amino acid transporter EAAT3Esslinger, C. Sean; Agarwal, Shailesh; Gerdes, John; Wilson, Paul A.; Davis, Erin S.; Awes, Alicia N.; O'Brien, Erin; Mavencamp, Teri; Koch, Hans P.; Poulsen, David J.; Rhoderick, Joseph F.; Chamberlin, A. Richard; Kavanaugh, Michael P.; Bridges, Richard J.Neuropharmacology (2005), 49 (6), 850-861CODEN: NEPHBW; ISSN:0028-3908. (Elsevier B.V.)The excitatory amino acid transporters (EAATs) play key roles in the regulation of CNS L-glutamate, esp. related to synthesis, signal termination, synaptic spillover, and excitotoxic protection. Inhibitors available to delineate EAAT pharmacol. and function are essentially limited to those that non-selectively block all EAATs or those that exhibit a substantial preference for EAAT2. Thus, it is difficult to selectively study the other subtypes, particularly EAAT1 and EAAT3. Structure activity studies on a series of β-substituted aspartate analogs identify L-β-benzyl-aspartate (L-β-BA) as among the first blockers that potently and preferentially inhibits the neuronal EAAT3 subtype. Kinetic anal. of D-[3H]aspartate uptake into C17.2 cells expressing the hEAATs demonstrate that L-β-threo-BA is the more potent diastereomer, acts competitively, and exhibits a 10-fold preference for EAAT3 compared to EAAT1 and EAAT2. Electrophysiol. recordings of EAAT-mediated currents in Xenopus oocytes identify L-β-BA as a non-substrate inhibitor. Analyzing L-β-threo-BA within the context of a novel EAAT2 pharmacophore model suggests: a highly conserved positioning of the electrostatic carboxyl and amino groups; nearby regions that accommodate select structural modifications (cyclopropyl rings, Me groups, oxygen atoms); and a unique region L-β-threo-BA occupied by the benzyl moieties of L-TBOA, L-β-threo-BA and related analogs. It is plausible that the preference of L-β-threo-BA and L-TBOA for EAAT3 and EAAT2, resp., could reside in the latter two pharmacophore regions.
- 46Greenfield, A.; Grosanu, C.; Dunlop, J.; McIlvain, B.; Carrick, T.; Jow, B.; Lu, Q.; Kowal, D.; Williams, J.; Butera, J. Synthesis and Biological Activities of Aryl-Ether-, Biaryl-, and Fluorene-Aspartic Acid and Diaminopropionic Acid Analogs as Potent Inhibitors of the High-Affinity Glutamate Transporter EAAT2. Bioorg. Med. Chem. Lett. 2005, 15, 4985– 4988, DOI: 10.1016/j.bmcl.2005.08.003Google Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtVynu7zM&md5=a3eab05582346ee6fb135d8134882d6fSynthesis and biological activities of aryl-ether-, biaryl-, and fluorene-aspartic acid and diaminopropionic acid analogs as potent inhibitors of the high-affinity glutamate transporter EAAT-2Greenfield, Alexander; Grosanu, Cristina; Dunlop, John; McIlvain, Beal; Carrick, Tikva; Jow, Brian; Lu, Qiang; Kowal, Dianne; Williams, John; Butera, JohnBioorganic & Medicinal Chemistry Letters (2005), 15 (22), 4985-4988CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)Excitatory amino acid transporters (EAATs) play a pivotal role in maintaining glutamate homeostasis in the mammalian central nervous system, with the EAAT-2 subtype thought to be responsible for the bulk of the glutamate uptake in forebrain regions. A complete elucidation of the functional role of EAAT-2 has been hampered by the lack of potent and selective pharmacol. tools. In this study, the authors describe the synthesis and biol. activities of novel aryl-ether, biaryl-, and fluorene-aspartic acid and diaminopropionic acid analogs as potent inhibitors of EAAT-2. Aspartate deriv. I represents one of the most potent (IC50 = 85 ± 5 nM) and selective inhibitors of EAAT-2 identified to date.
- 47Jensen, A. A.; Erichsen, M. N.; Nielsen, C. W.; Stensbøl, T. B.; Kehler, J.; Bunch, L. Discovery of the First Selective Inhibitor of Excitatory Amino Acid Transporter Subtype 1. J. Med. Chem. 2009, 52 (4), 912– 915, DOI: 10.1021/jm8013458Google Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtVSqs78%253D&md5=7036a6fe617a9908a36fda6a87400e2cDiscovery of the First Selective Inhibitor of Excitatory Amino Acid Transporter Subtype 1Jensen, Anders A.; Erichsen, Mette N.; Nielsen, Christina W.; Stensboel, Tine B.; Kehler, Jan; Bunch, LennartJournal of Medicinal Chemistry (2009), 52 (4), 912-915CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The discovery of the first class of subtype-selective inhibitors of the human excitatory amino acid transporter subtype 1 (EAAT1) and its rat orthologue GLAST is reported. An opening structure-activity relationship of 25 analogs is presented that addresses the influence of substitutions at the 4- and 7-positions of the parental skeleton 2-amino-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile. The most potent analog 10 displays high nanomolar inhibitory activity at EAAT1 and a >400-fold selectivity over EAAT2 and EAAT3, making it a highly valuable pharmacol. tool.
- 48Hansen, S. W.; Erichsen, M. N.; Fu, B.; Bjørn-Yoshimoto, W. E.; Abrahamsen, B.; Hansen, J. C.; Jensen, A. A.; Bunch, L. Identification of a New Class of Selective Excitatory Amino Acid Transporter Subtype 1 (EAAT1) Inhibitors Followed by a Structure-Activity Relationship Study. J. Med. Chem. 2016, 59 (19), 8757– 8770, DOI: 10.1021/acs.jmedchem.6b01058Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsFSjurnK&md5=2151faee0204fe3faff7b003950628dcIdentification of a New Class of Selective Excitatory Amino Acid Transporter Subtype 1 (EAAT1) Inhibitors Followed by a Structure-Activity Relationship StudyHansen, Stinne W.; Erichsen, Mette N.; Fu, Bingru; Bjoern-Yoshimoto, Walden E.; Abrahamsen, Bjarke; Hansen, Jacob C.; Jensen, Anders A.; Bunch, LennartJournal of Medicinal Chemistry (2016), 59 (19), 8757-8770CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Screening of a small compd. library at three excitatory amino acid transporter subtypes 1-3 (EAAT1-3) subtypes resulted in the identification of compd. (I) that exhibited a distinct preference as an inhibitor at EAAT1 (IC50 20 μM) compared to EAAT2 and EAAT3 (IC50 > 300 μM). This prompted us to subject I to an elaborate structure-activity-relationship study through the purchase and synthesis and subsequent pharmacol. characterization of a total of 36 analogs. Although this effort did not result in analogs with substantially improved inhibitory potencies at EAAT1 compared to that displayed by the hit, it provided a detailed insight into structural requirements for EAAT1 activity of this scaffold. The discovery of this new class of EAAT1-selective inhibitors not only supplements the currently available pharmacol. tools in the EAAT field, but also substantiates the notion that EAAT ligands not derived from α-amino acids hold considerable potential in terms of subtype-selective modulation.
- 49Abrahamsen, B.; Schneider, N.; Erichsen, M. N.; Huynh, T. H. V.; Fahlke, C.; Bunch, L.; Jensen, A. A. Allosteric Modulation of an Excitatory Amino Acid Transporter: The Subtype-Selective Inhibitor UCPH-101 Exerts Sustained Inhibition of EAAT1 through an Intramonomeric Site in the Trimerization Domain. J. Neurosci. 2013, 33 (3), 1068– 1087, DOI: 10.1523/JNEUROSCI.3396-12.2013Google Scholar49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsVemsLg%253D&md5=5e2b79367256a04008e6180cb9856d6fAllosteric modulation of an excitatory amino acid transporter:the subtype-selective inhibitor UCPH-101 exerts sustained inhibition of EAAT1 through an intramonomeric site in the trimerization domainAbrahamsen, Bjarke; Schneider, Nicole; Erichsen, Mette N.; Huynh, Tri H. V.; Fahlke, Christoph; Bunch, Lennart; Jensen, Anders A.Journal of Neuroscience (2013), 33 (3), 1068-1087CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)In the present study, the mechanism of action and mol. basis for the activity of the first class of selective inhibitors of the human excitatory amino acid transporter subtype 1 (EAAT1) and its rodent ortholog GLAST are elucidated. The previously reported specificity of UCPH-101 and UCPH-102 for EAAT1 over EAAT2 and EAAT3 is demonstrated to extend to the EAAT4 and EAAT5 subtypes as well. Interestingly, brief exposure to UCPH-101 induces a long-lasting inactive state of EAAT1, whereas the inhibition exerted by closely related analogs is substantially more reversible in nature. In agreement with this, the kinetic properties of UCPH-101 unblocking of the transporter are considerably slower than those of UCPH-102. UCPH-101 exhibits noncompetitive inhibition of EAAT1, and its binding site in GLAST has been delineated in an elaborate mutagenesis study. Substitutions of several residues in TM3, TM4c, and TM7a of GLAST have detrimental effects on the inhibitory potency and/or efficacy of UCPH-101 while not affecting the pharmacol. properties of (S)-glutamate or the competitive EAAT inhibitor TBOA significantly. Hence, UCPH-101 is proposed to target a predominantly hydrophobic crevice in the "trimerization domain" of the GLAST monomer, and the inhibitor is demonstrated to inhibit the uptake through the monomer that it binds to exclusively and not to affect substrate translocation through the other monomers in the GLAST trimer. The allosteric mode of UCPH-101 inhibition underlines the functional importance of the trimerization domain of the EAAT and demonstrates the feasibility of modulating transporter function through ligand binding to regions distant from its "transport domain.".
- 50Balcar, V. J.; Johnston, G. A. R. The Structural Specificity of the High Affinity Uptake of L-Glutamate and L-Aspartate by Rat Brain Slices. J. Neurochem. 1972, 19 (11), 2657– 2666, DOI: 10.1111/j.1471-4159.1972.tb01325.xGoogle Scholar50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE3sXmslKhtw%253D%253D&md5=3a14f9449d9764ca8f1ee9f48d8532b4Structural specificity of the high-affinity uptake of L-glutamate and L-aspartate by rat brain slicesBalcar, V. J.; Johnston, G. A. R.Journal of Neurochemistry (1972), 19 (11), 2657-66CODEN: JONRA9; ISSN:0022-3042.The high-affinity uptake system for L-glutamate [56-86-0] and L-aspartate [56-84-8] in rat cerebral cortex may not be specific for these likely excitatory synaptic transmitters, since threo-3-hydroxy-DL-aspartate [4294-45-5], L-cysteinesulfinate [15932-83-9], L-cysteate [498-40-8], and D-aspartate [1783-96-6] strongly inhibited the obsd. high-affinity uptake of L-glutamate by rat brain slices in a manner consistent with linear competitive inhibition. L-glutamate high-affinity uptake was Na+-dependent, but under certain conditions appeared to be less sensitive than γ-aminobutyrate uptake to changes in the external Na+ concn. and to drugs which modify Na+ movements. This may be relevant to the efficiency of the glutamate uptake process during synaptic depolarization induced by glutamate. L-glutamate high-affinity uptake was also inhibited in a relatively nonspecific manner by a variety of drugs including mercurials and some electron transport inhibitors.
- 51Shimamoto, K.; LeBrun, B.; Yasuda-Kamatani, Y.; Sakaitani, M.; Shigeri, Y.; Yumoto, N.; Nakajima, T. DL-Threo-Beta-Benzyloxyaspartate, a Potent Blocker of Excitatory Amino Acid Transporters. Mol. Pharmacol. 1998, 53, 195– 201, DOI: 10.1124/mol.53.2.195Google Scholar51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXht12htrw%253D&md5=90eb235209edbbcaa9bf5a7e69dae743DL-threo-β-Benzyloxyaspartate, a potent blocker of excitatory amino acid transportersShimamoto, Keiko; Lebrun, Bruno; Yasuda-Kamatani, Yoshimi; Sakaitani, Masahiro; Shigeri, Yasushi; Yumoto, Noboru; Nakajima, TerumiMolecular Pharmacology (1998), 53 (2), 195-201CODEN: MOPMA3; ISSN:0026-895X. (Williams & Wilkins)DL-Threo-β-Benzyloxyaspartate (DL-TBOA), a novel deriv. of DL-threo-β-hydroxyaspartate, was synthesized and examd. as an inhibitor of sodium-dependent glutamate/aspartate (excitatory amino acid) transporters. DL-TBOA inhibited the uptake of [14C]glutamate in COS-1 cells expressing the human excitatory amino acid transporter-1 (EAAT1) (Ki = 42 μM) with almost the same potency as DL-threo-β-hydroxyaspartate (Ki = 58 μM). With regard to the human excitatory amino acid transporter-2 (EAAT2), the inhibitory effect of DL-TBOA (Ki = 5.7 μM) was much more potent than that of dihydrokainate (Ki = 79 μM), which is well known as a selective blocker of this subtype. Electrophysiol., DL-TBOA induced no detectable inward currents in Xenopus laevis oocytes expressing human EAAT1 or EAAT2. However, it significantly reduced the glutamate-induced currents, indicating the prevention of transport. The dose-response curve of glutamate was shifted by adding DL-TBOA without a significant change in the max. current. The Kb values for human EAAT1 and EAAT2 expressed in X. laevis oocytes were 9.0 μM and 116 nM, resp. These results demonstrated that DL-TBOA is, so far, the most potent competitive blocker of glutamate transporters. DL-TBOA did not show any significant effects on either the ionotropic or metabotropic glutamate receptors. Moreover, DL-TBOA is chem. much more stable than its benzoyl analog, a previously reported blocker of excitatory amino acid transporters; therefore, DL-TBOA should be a useful tool for investigating the physiol. roles of transporters.
- 52Shimamoto, K.; Sakai, R.; Takaoka, K.; Yumoto, N.; Nakajima, T.; Amara, S. G.; Shigeri, Y. Characterization of Novel L-Threo-Beta-Benzyloxyaspartate Derivatives, Potent Blockers of the Glutamate Transporters. Mol. Pharmacol. 2004, 65 (4), 1008– 1015, DOI: 10.1124/mol.65.4.1008Google Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXivFaltbo%253D&md5=48cc0d672b697001a4161172920406e8Characterization of novel L-threo-β-benzyloxyaspartate derivatives, potent blockers of the glutamate transportersShimamoto, Keiko; Sakai, Ryuichi; Takaoka, Kiyo; Yumoto, Noboru; Nakajima, Terumi; Amara, Susan G.; Shigeri, YasushiMolecular Pharmacology (2004), 65 (4), 1008-1015CODEN: MOPMA3; ISSN:0026-895X. (American Society for Pharmacology and Experimental Therapeutics)Nontransportable blockers of the glutamate transporters are important tools for investigating mechanisms of synaptic transmission. DL-threo-β-Benzyloxyaspartate (DL-TBOA) is a potent blocker of all subtypes of the excitatory amino acid transporters (EAATs). We characterized novel L-TBOA analogs possessing a substituent on their resp. benzene rings. The analogs significantly inhibited labeled glutamate uptake, the most potent of which was (2S,3S)-3-{3-[4-(trifluoromethyl)benzoylamino]benzyloxy}aspartate (TFB-TBOA). In an uptake assay using cells transiently expressing EAATs, the IC50 values of TFB-TBOA for EAAT1, EAAT2, and EAAT3 were 22, 17, and 300 nM, resp. TFB-TBOA was significantly more potent at inhibiting EAAT1 and EAAT2 compared with L-TBOA (IC50 values for EAAT1-3 were 33, 6.2, and 15 μM, resp.). Electrophysiol. analyses revealed that TBOA analogs block the transport-assocd. currents in all five EAAT subtypes and also block leak currents in EAAT5. The rank order of the analogs for potencies at inhibiting substrate-induced currents was identical to that obsd. in the uptake assay. However, the kinetics of TFB-TBOA differed from the kinetics of L-TBOA, probably because of the strong binding affinity. Notably, TFB-TBOA did not affect other representative neurotransmitter transporters or receptors, including ionotropic and metabotropic glutamate receptors, indicating that it is highly selective for EAATs. Moreover, intracerebroventricular administration of the TBOA analogs induced severe convulsive behaviors in mice, probably because of the accumulation of glutamate. Taken together, these findings indicate that novel TBOA analogs, esp. TFB-TBOA, should serve as useful tools for elucidating the physiol. roles of the glutamate transporters.
- 53Fu, H.; Zhang, J.; Tepper, P. G.; Bunch, L.; Jensen, A. A.; Poelarends, G. J. Chemoenzymatic Synthesis and Pharmacological Characterization of Functionalized Aspartate Analogues as Novel Excitatory Amino Acid Transporter Inhibitors. J. Med. Chem. 2018, 61 (17), 7741– 7753, DOI: 10.1021/acs.jmedchem.8b00700Google Scholar53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtlantb3E&md5=7cc9859a2743c381856009492c23de2cChemoenzymatic Synthesis and Pharmacological Characterization of Functionalized Aspartate Analogues As Novel Excitatory Amino Acid Transporter InhibitorsFu, Haigen; Zhang, Jielin; Tepper, Pieter G.; Bunch, Lennart; Jensen, Anders A.; Poelarends, Gerrit J.Journal of Medicinal Chemistry (2018), 61 (17), 7741-7753CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Aspartate (Asp) derivs. are privileged compds. for investigating the roles governed by excitatory amino acid transporters (EAATs) in glutamatergic neurotransmission. Here, we report the synthesis of various Asp derivs. with (cyclo)alkyloxy and (hetero)aryloxy substituents at C-3. Their pharmacol. properties were characterized at the EAAT1-4 subtypes. The L-threo-3-substituted Asp derivs. (I) (R1 = cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, 2-thiophenyl, 3-thiophenyl, 2-furanyl, 3-furanyl, propargyl) were non-substrate inhibitors, exhibiting pan activity at EAAT1-4 with IC50 values ranging from 0.49 to 15 μM. Comparisons between (DL-threo)- and (DL-erythro)-Asp analogs I (R1 = cyclopropyl, 3-thiophenyl, propargyl) confirmed that the threo configuration is crucial for the EAAT1-4 inhibitory activities. Analogs I (R1 = cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl) of L-TFB-TBOA I (R1 = cyclopropyl) were shown to be potent EAAT1-4 inhibitors, with IC50 values ranging from 5-530 nM. Hybridization of the nonselective EAAT inhibitor L-TBOA with EAAT2-selective inhibitor WAY-213613 or EAAT3-preferring inhibitor NBI-59159 yielded compds. (II)(R2 = III and IV) resp., which were non-selective EAAT inhibitors displaying considerably lower IC50 values at EAAT1-4 (11-140 nM) than those displayed by the resp. parent mols.
- 54Canul-Tec, J. C.; Assal, R.; Cirri, E.; Legrand, P.; Brier, S.; Chamot-Rooke, J.; Reyes, N. Structure and Allosteric Inhibition of Excitatory Amino Acid Transporter 1. Nature 2017, 544, 446– 451, DOI: 10.1038/nature22064Google Scholar54https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmtlalt7k%253D&md5=1107e965f53e44801b6f51152f29ba13Structure and allosteric inhibition of excitatory amino acid transporter 1Canul-Tec, Juan C.; Assal, Reda; Cirri, Erica; Legrand, Pierre; Brier, Sebastien; Chamot-Rooke, Julia; Reyes, NicolasNature (London, United Kingdom) (2017), 544 (7651), 446-451CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Human members of the solute carrier 1 (SLC1) family of transporters take up excitatory neurotransmitters in the brain and amino acids in peripheral organs. Dysregulation of the function of SLC1 transporters is assocd. with neurodegenerative disorders and cancer. Here we present crystal structures of a thermostabilized human SLC1 transporter, the excitatory amino acid transporter 1 (EAAT1), with and without allosteric and competitive inhibitors bound. The structures reveal architectural features of the human transporters, such as intra- and extracellular domains that have potential roles in transport function, regulation by lipids and post-translational modifications. The coordination of the allosteric inhibitor in the structures and the change in the transporter dynamics measured by hydrogen-deuterium exchange mass spectrometry reveal a mechanism of inhibition, in which the transporter is locked in the outward-facing states of the transport cycle. Our results provide insights into the mol. mechanisms underlying the function and pharmacol. of human SLC1 transporters.
- 55Garaeva, A. A.; Guskov, A.; Slotboom, D. J.; Paulino, C. A One-Gate Elevator Mechanism for the Human Neutral Amino Acid Transporter ASCT2. Nat. Commun. 2019, 10 (1), 3427, DOI: 10.1038/s41467-019-11363-xGoogle Scholar55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3MvjsVensg%253D%253D&md5=bdb8e27fd66ac8d3a59fc28b3740d9d1A one-gate elevator mechanism for the human neutral amino acid transporter ASCT2Garaeva Alisa A; Slotboom Dirk J; Paulino Cristina; Guskov Albert; Paulino Cristina; Slotboom Dirk JNature communications (2019), 10 (1), 3427 ISSN:.The human Alanine Serine Cysteine Transporter 2 (ASCT2) is a neutral amino acid exchanger that belongs to the solute carrier family 1 (SLC1A). SLC1A structures have revealed an elevator-type mechanism, in which the substrate is translocated across the cell membrane by a large displacement of the transport domain, whereas a small movement of hairpin 2 (HP2) gates the extracellular access to the substrate-binding site. However, it has remained unclear how substrate binding and release is gated on the cytoplasmic side. Here, we present an inward-open structure of the human ASCT2, revealing a hitherto elusive SLC1A conformation. Strikingly, the same structural element (HP2) serves as a gate in the inward-facing as in the outward-facing state. The structures reveal that SLC1A transporters work as one-gate elevators. Unassigned densities near the gate and surrounding the scaffold domain, may represent potential allosteric binding sites, which could guide the design of lipidic-inhibitors for anticancer therapy.
- 56Cheng, B.; Trauner, D.; Shchepakin, D.; Kavanaugh, M. P. A Photoswitchable Inhibitor of a Glutamate Transporter. ACS Chem. Neurosci. 2017, 8, 1668– 1672, DOI: 10.1021/acschemneuro.7b00072Google Scholar56https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmtFCmuro%253D&md5=7a430f138b3da62ba24ff4ceb2f7a681Photoswitchable Inhibitor of a Glutamate TransporterCheng, Bichu; Shchepakin, Denis; Kavanaugh, Michael P.; Trauner, DirkACS Chemical Neuroscience (2017), 8 (8), 1668-1672CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)Excitatory amino acid transporters clear glutamate from the synaptic cleft and play a crit. role in glutamatergic neurotransmission. Their differential roles in astrocytes, microglia, and neurons are poorly understood due in part to a lack of pharmacol. tools that can be targeted to specific cells and tissues. We now describe a photoswitchable inhibitor, termed ATT, that interacts with the major mammalian forebrain transporters EAAT1-3 in a manner that can be reversibly switched between trans (high-affinity) and cis (low-affinity) configurations using light of different colors. In the dark, ATT competitively inhibited the predominant glial transporter EAAT2 with ∼200-fold selectivity over the neuronal transporter EAAT3. Brief exposure to 350 nm light reduced the steady-state blocker affinity by more than an order of magnitude. Illumination of EAAT2 complexed with ATT induced a corresponding increase in the blocker off-rate monitored in the presence of glutamate. ATT can be used to reversibly manipulate glutamate transporter activity with light and may be useful to gain insights into the dynamic physiol. roles of glutamate transporters in the brain, as well as to study the mol. interactions of transporters with ligands.
- 57Hoorens, M. W. H.; Fu, H.; Duurkens, R. H.; Trinco, G.; Arkhipova, V.; Feringa, B. L.; Poelarends, G. J.; Slotboom, D. J.; Szymanski, W. Glutamate Transporter Inhibitors with Photo-Controlled Activity. Adv. Ther. 2018, 1, 1800028, DOI: 10.1002/adtp.201800028Google ScholarThere is no corresponding record for this reference.
- 58Foster, A. C.; Li, Y.-X.; Runyan, S.; Dinh, T.; Venadas, S.; Chen, J.; Pashikanti, S.; Datta, A.; Ehring, G.; Staubli, U. Activity of the Enantiomers of Erythro-3-Hydroxyaspartate at Glutamate Transporters and NMDA Receptors. J. Neurochem. 2016, 136, 692– 697, DOI: 10.1111/jnc.13430Google Scholar58https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXitVemtLnE&md5=0e9c224d70b60bf40cab4f0133c5ebbdActivity of the enantiomers of erythro-3-hydroxyaspartate at glutamate transporters and NMDA receptorsFoster, Alan C.; Li, Yong-Xin; Runyan, Stephen; Dinh, Tim; Venadas, Steven; Chen, June; Pashikanti, Srinath; Datta, Apurba; Ehring, George; Staubli, UrsulaJournal of Neurochemistry (2016), 136 (4), 692-697CODEN: JONRA9; ISSN:0022-3042. (Wiley-Blackwell)The enantiomers of erythro-3-hydroxyaspartate were tested for activity at glutamate transporters and NMDA receptors. Both enantiomers inhibited glutamate transporters in rat hippocampal crude synaptosomes and elicited substrate-like activity at excitatory amino acid transporter 1, 2, and 3 as measured by voltage clamp in the Xenopus oocyte expression system. The enantiomers had similar affinities, but the D-enantiomer showed a lower maximal effect at excitatory amino acid transporter 1, 2, and 3 than the L-enantiomer. Surprisingly, D-erythro-3-hydroxyaspartate was a potent NMDA receptor agonist with an EC50 value in rat hippocampal neurons of 320 nM, whereas the L-enantiomer was 100-fold less potent. L-erythro-3-hydroxyaspartate showed activity at both glutamate transporters and NMDA receptors at concns. that are reported to inhibit serine racemase, indicating a lack of selectivity. This enantiomeric pair may assist in shedding further light on the structural requirements for substrate activity at glutamate transporters and for agonist activity at NMDA receptors. The erythro enantiomers of 3-hydroxyaspartate had interesting and surprising effects on glutamate neurotransmitter systems. L-erythro-3-hydroxyaspartate had activity at both glutamate transporters (EAAT1/2/3) and NMDA receptors. D-erythro-3-hydroxyaspartate acted on EAATs, but was also identified as a highly potent NMDA receptor agonist. These enantiomers shed further light on the structural requirements for activity at EAATs and NMDA receptors.
- 59Tolner, B.; Ubbink-Kok, T.; Poolman, B.; Konings, W. N. Characterization of the Proton/Glutamate Symport Protein of Bacillus Subtilis and Its Functional Expression in Escherichia Coli. J. Bacteriol. 1995, 177 (10), 2863– 2869, DOI: 10.1128/JB.177.10.2863-2869.1995Google Scholar59https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXls1Sks7w%253D&md5=a96a4474c5a9f3fd1c07837b8dadfb72Characterization of the proton/glutamate symport protein of Bacillus subtilis and its functional expression in Escherichia coliTolner, Berend; Ubbink-Kok, Trees; Poolman, Bert; Konings, Wil N.Journal of Bacteriology (1995), 177 (10), 2863-9CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)Transport of acidic amino acids in Bacillus subtilis is an electrogenic process in which L-glutamate or L-aspartate is symported with at least two protons. This is shown by studies of transport in membrane vesicles in which a proton motive force is generated by oxidn. of ascorbate-phenazine methosulfate or by artificial ion gradients. An inwards-directed sodium gradient had no (stimulatory) effect on proton motive force-driven L-glutamate uptake. The transporter is specific for L-glutamate and L-aspartate. L-Glutamate transport is inhibited by β-hydroxyaspartate and cysteic acid but not by α-methyl-glutamate. The gene encoding the L-glutamate transport protein of B. subtilis (gltPBsu) was cloned by complementation of Escherichia coli JC5412 for growth on glutamate as the sole source of carbon, energy, and nitrogen, and its nucleotide sequence was detd. Putative promoter, terminator, and ribosome binding site sequences were found in the flanking regions. UUG is most likely the start codon. GltPBsu encodes a polypeptide of 414 amino acid residues and is homologous to several proteins that transport glutamate and/or structurally related compds. such as aspartate, fumarate, malate, and succinate. Both sodium- and proton-coupled transporters belong to this family of dicarboxylate transporters. Hydropathy profiling and multiple alignment of the fatty of carboxylate transporters suggest that each of the proteins spans the cytoplasmic membrane 12 times with both the amino and carboxy termini on the inside.
- 60Tolner, B.; Ubbink-Kok, T.; Poolmann, B.; Konings, W. N. Cation-Selectivity of the L-Glutamate Transporters of Escherichia Coli, Bacillus Stearothermophilus and Bacillus Caldotenax: Dependence on the Environment in Which the Proteins Are Expressed. Mol. Microbiol. 1995, 18 (1), 123– 133, DOI: 10.1111/j.1365-2958.1995.mmi_18010123.xGoogle Scholar60https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXptlersbw%253D&md5=54084c9847f378f805022850afd0122cCation-selectivity of the L-glutamate transporters of Escherichia coli, Bacillus stearothermophilus and Bacillus caldotenax: dependence on the environment in which the proteins are expressedTolner, Berend; Ubbink-Kok, Trees; Poolman, Bert; Konings, Will N.Molecular Microbiology (1995), 18 (1), 123-33CODEN: MOMIEE; ISSN:0950-382X. (Blackwell)L-Glutamate transport by the H+-glutamate and Na+-glutamate symport proteins of Escherichia coli K-12 (GltPEc and GltSEc, resp.) and the Na+-H+-glutamate symport proteins of Bacillus stearothermophilus (GltTBs) and Bacillus caldotenax (GltTBc) was studied in membrane vesicles derived from cells in which the proteins were either homologously or heterologously expressed. Substrate and inhibitor specificity studies indicate that GltPEc, GltTBs and GltTBc fall into the same group of transporters, whereas GltSEc is distinctly different from the others. Also, the cation specificity of GltSEc is different; GltSEc transported L-glutamate with (at least) two Na+, whereas GltPEc, GltTBs and GltTBc catalyzed an electrogenic symport of L-glutamate with ≥two H+, i.e. when the proteins were expressed in E. coli. Surprisingly studies in membrane vesicles of B. stearothermophilus and B. caldotenax indicated a Na+-H+-L-glutamate symport for both GltTBs and GltTBc. The Na+ dependency of the GltT transporters in the Bacillus strains increased with temp. These observations suggest that the conformation of the transport proteins in the E. coli and the Bacillus membranes differs, which influences the coupling ion selectivity.
- 61Woodhouse, J.; Nass Kovacs, G.; Coquelle, N.; Uriarte, L. M.; Adam, V.; Barends, T. R. M.; Byrdin, M.; de la Mora, E.; Bruce Doak, R.; Feliks, M.; Field, M.; Fieschi, F.; Guillon, V.; Jakobs, S.; Joti, Y.; Macheboeuf, P.; Motomura, K.; Nass, K.; Owada, S.; Roome, C. M.; Ruckebusch, C.; Schirò, G.; Shoeman, R. L.; Thepaut, M.; Togashi, T.; Tono, K.; Yabashi, M.; Cammarata, M.; Foucar, L.; Bourgeois, D.; Sliwa, M.; Colletier, J.-P.; Schlichting, I.; Weik, M. Photoswitching Mechanism of a Fluorescent Protein Revealed by Time-Resolved Crystallography and Transient Absorption Spectroscopy. Nat. Commun. 2020, 11, 741, DOI: 10.1038/s41467-020-14537-0Google Scholar61https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXjs1OhtLY%253D&md5=1564ff90993aa05bf9fad976808fc853Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopyWoodhouse, Joyce; Nass Kovacs, Gabriela; Coquelle, Nicolas; Uriarte, Lucas M.; Adam, Virgile; Barends, Thomas R. M.; Byrdin, Martin; de la Mora, Eugenio; Bruce Doak, R.; Feliks, Mikolaj; Field, Martin; Fieschi, Franck; Guillon, Virginia; Jakobs, Stefan; Joti, Yasumasa; Macheboeuf, Pauline; Motomura, Koji; Nass, Karol; Owada, Shigeki; Roome, Christopher M.; Ruckebusch, Cyril; Schiro, Giorgio; Shoeman, Robert L.; Thepaut, Michel; Togashi, Tadashi; Tono, Kensuke; Yabashi, Makina; Cammarata, Marco; Foucar, Lutz; Bourgeois, Dominique; Sliwa, Michel; Colletier, Jacques-Philippe; Schlichting, Ilme; Weik, MartinNature Communications (2020), 11 (1), 741CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Reversibly switchable fluorescent proteins (RSFPs) serve as markers in advanced fluorescence imaging. Photoswitching from a non-fluorescent off-state to a fluorescent on-state involves trans-to-cis chromophore isomerization and proton transfer. Whereas excited-state events on the ps timescale have been structurally characterized, conformational changes on slower timescales remain elusive. Here we describe the off-to-on photoswitching mechanism in the RSFP rsEGFP2 by using a combination of time-resolved serial crystallog. at an X-ray free-electron laser and ns-resolved pump-probe UV-visible spectroscopy. Ten ns after photoexcitation, the crystal structure features a chromophore that isomerized from trans to cis but the surrounding pocket features conformational differences compared to the final on-state. Spectroscopy identifies the chromophore in this ground-state photo-intermediate as being protonated. Deprotonation then occurs on the μs timescale and correlates with a conformational change of the conserved neighboring histidine. Together with a previous excited-state study, our data allow establishing a detailed mechanism of off-to-on photoswitching in rsEGFP2.
- 62Fu, H.; Younes, S. H. H.; Saifuddin, M.; Tepper, P. G.; Zhang, J.; Keller, E.; Heeres, A.; Szymanski, W.; Poelarends, G. J. Rapid Chemoenzymatic Route to Glutamate Transporter Inhibitor L-TFB-TBOA and Related Amino Acids. Org. Biomol. Chem. 2017, 15 (11), 2341– 2344, DOI: 10.1039/C7OB00305FGoogle Scholar62https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXjtFehu7k%253D&md5=f66db63d5a1f2e026f7b8a49b0e31c48Rapid chemoenzymatic route to glutamate transporter inhibitor L-TFB-TBOA and related amino acidsFu, Haigen; Younes, Sabry H. H.; Saifuddin, Mohammad; Tepper, Pieter G.; Zhang, Jielin; Keller, Erik; Heeres, Andre; Szymanski, Wiktor; Poelarends, Gerrit J.Organic & Biomolecular Chemistry (2017), 15 (11), 2341-2344CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)The complex amino acid (L-threo)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (L-TFB-TBOA) and its derivs. are privileged compds. for studying the roles of excitatory amino acid transporters (EAATs) in regulation of glutamatergic neurotransmission, animal behavior, and in the pathogenesis of neurol. diseases. The wide-spread use of L-TFB-TBOA stems from its high potency of EAAT inhibition and the lack of off-target binding to glutamate receptors. However, one of the main challenges in the evaluation of L-TFB-TBOA and its derivs. is the laborious synthesis of these compds. in stereoisomerically pure form. Here, we report an efficient and step-economic chemoenzymic route that gives access to enantio- and diastereopure L-TFB-TBOA and its derivs. at multigram scale.
- 63Raj, H.; Szymański, W.; De Villiers, J.; Rozeboom, H. J.; Veetil, V. P.; Reis, C. R.; De Villiers, M.; Dekker, F. J.; De Wildeman, S.; Quax, W. J.; Thunnissen, A. M. W. H.; Feringa, B. L.; Janssen, D. B.; Poelarends, G. J. Engineering Methylaspartate Ammonia Lyase for the Asymmetric Synthesis of Unnatural Amino Acids. Nat. Chem. 2012, 4 (6), 478– 484, DOI: 10.1038/nchem.1338Google Scholar63https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xmt1Gktb8%253D&md5=e8fc35523cd38b2cb130ac8fc4813b1bEngineering methylaspartate ammonia lyase for the asymmetric synthesis of unnatural amino acidsRaj, Hans; Szymanski, Wiktor; de Villiers, Jandre; Rozeboom, Henriette J.; Veetil, Vinod Puthan; Reis, Carlos R.; de Villiers, Marianne; Dekker, Frank J.; de Wildeman, Stefaan; Quax, Wim J.; Thunnissen, Andy-Mark W. H.; Feringa, Ben L.; Janssen, Dick B.; Poelarends, Gerrit J.Nature Chemistry (2012), 4 (6), 478-484CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)The redesign of enzymes to produce catalysts for a predefined transformation remains a major challenge in protein engineering. Here, we describe the structure-based engineering of methylaspartate ammonia lyase (which in nature catalyzes the conversion of 3-methylaspartate to ammonia and 2-methylfumarate) to accept a variety of substituted amines and fumarates and catalyze the asym. synthesis of aspartic acid derivs. We obtained two single-active-site mutants, one exhibiting a wide nucleophile scope including structurally diverse linear and cyclic alkylamines and one with broad electrophile scope including fumarate derivs. with alkyl, aryl, alkoxy, aryloxy, alkylthio and arylthio substituents at the C2 position. Both mutants have an enlarged active site that accommodates the new substrates while retaining the high stereo- and regioselectivity of the wild-type enzyme. As an example, we demonstrate a highly enantio- and diastereoselective synthesis of threo-3-benzyloxyaspartate (an important inhibitor of neuronal excitatory glutamate transporters in the brain).
- 64Cheng, B.; Shchepakin, D.; Kavanaugh, M. P.; Trauner, D. Photoswitchable Inhibitor of a Glutamate Transporter. ACS Chem. Neurosci. 2017, 8 (8), 1668– 1672, DOI: 10.1021/acschemneuro.7b00072Google Scholar64https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmtFCmuro%253D&md5=7a430f138b3da62ba24ff4ceb2f7a681Photoswitchable Inhibitor of a Glutamate TransporterCheng, Bichu; Shchepakin, Denis; Kavanaugh, Michael P.; Trauner, DirkACS Chemical Neuroscience (2017), 8 (8), 1668-1672CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)Excitatory amino acid transporters clear glutamate from the synaptic cleft and play a crit. role in glutamatergic neurotransmission. Their differential roles in astrocytes, microglia, and neurons are poorly understood due in part to a lack of pharmacol. tools that can be targeted to specific cells and tissues. We now describe a photoswitchable inhibitor, termed ATT, that interacts with the major mammalian forebrain transporters EAAT1-3 in a manner that can be reversibly switched between trans (high-affinity) and cis (low-affinity) configurations using light of different colors. In the dark, ATT competitively inhibited the predominant glial transporter EAAT2 with ∼200-fold selectivity over the neuronal transporter EAAT3. Brief exposure to 350 nm light reduced the steady-state blocker affinity by more than an order of magnitude. Illumination of EAAT2 complexed with ATT induced a corresponding increase in the blocker off-rate monitored in the presence of glutamate. ATT can be used to reversibly manipulate glutamate transporter activity with light and may be useful to gain insights into the dynamic physiol. roles of glutamate transporters in the brain, as well as to study the mol. interactions of transporters with ligands.
- 65Morstein, J.; Awale, M.; Reymond, J. L.; Trauner, D. Mapping the Azolog Space Enables the Optical Control of New Biological Targets. ACS Cent. Sci. 2019, 5 (4), 607– 618, DOI: 10.1021/acscentsci.8b00881Google Scholar65https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXks1Witro%253D&md5=4a799a927450bddb1770f7801282ae94Mapping the Azolog Space Enables the Optical Control of New Biological TargetsMorstein, Johannes; Awale, Mahendra; Reymond, Jean-Louis; Trauner, DirkACS Central Science (2019), 5 (4), 607-618CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)Photopharmacol. relies on mols. that change their biol. activity upon irradn. Many of these are derived from known drugs by replacing their core with an isosteric azobenzene photoswitch (azologization). The question is how many of the known bioactive ligands could be addressed in such a way. Here, the authors systematically assess the space of mols. amenable to azologization from databases of bioactive mols. (DrugBank, PDB, CHEMBL) and the Cambridge Structural Database. Shape similarity scoring functions (3DAPfp) and analyses of dihedral angles were employed to quantify the structural homol. between a bioactive mol. and the cis or trans isomer of its corresponding azolog ("azoster") and assess which isomer is likely to be active. The authors' anal. suggests that a very large no. of bioactive ligands (>40,000) is amenable to azologization and that many new biol. targets could be addressed with photopharmacol. N-Aryl benzamides, 1,2-diarylethanes, and benzyl Ph ethers are particularly suited for this approach, while benzylanilines and sulfonamides appear to be less well-matched. On the basis of the authors' anal., the majority of azosters are expected to be active in their trans form. The broad applicability of the authors' approach is demonstrated with photoswitches that target a nuclear hormone receptor (RAR) and a lipid processing enzyme (LTA4 hydrolase).
- 66Hoorens, M. W. H.; Ourailidou, M. E.; Rodat, T.; van der Wouden, P. E.; Kobauri, P.; Kriegs, M.; Peifer, C.; Feringa, B. L.; Dekker, F. J.; Szymanski, W. Light-Controlled Inhibition of BRAFV600E Kinase. Eur. J. Med. Chem. 2019, 179, 133– 146, DOI: 10.1016/j.ejmech.2019.06.042Google Scholar66https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXht1Oqt7nI&md5=8fd9228db18dba5bb1a0a47f92aad8e7Light-controlled inhibition of BRAFV600E kinaseHoorens, Mark W. H.; Ourailidou, Maria E.; Rodat, Theo; van der Wouden, Petra E.; Kobauri, Piermichele; Kriegs, Malte; Peifer, Christian; Feringa, Ben L.; Dekker, Frank J.; Szymanski, WiktorEuropean Journal of Medicinal Chemistry (2019), 179 (), 133-146CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)Metastatic melanoma is amongst the most difficult types of cancer to treat, with current therapies mainly relying on the inhibition of the BRAFV600E mutant kinase. However, systemic inhibition of BRAF by small mol. drugs in cancer patients results - paradoxically - in increased wild-type BRAF activity in healthy tissue, causing side-effects and even the formation of new tumors. Here we show the development of BRAFV600E kinase inhibitors of which the activity can be switched on and off reversibly with light, offering the possibility to overcome problems of systemic drug activity by selectively activating the drug at the desired site of action. Based on a known inhibitor, eight photoswitchable effectors contg. an azobenzene photoswitch were designed, synthesized and evaluated. The most promising inhibitor showed an approx. 10-fold increase in activity upon light-activation. This research offers inspiration for the development of therapies for metastatic melanoma in which tumor tissue is treated with an active BRAFV600E inhibitor with high spatial and temporal resoln., thus limiting the damage to other tissues.
- 67Xu, Z.; Shi, L.; Jiang, D.; Cheng, J.; Shao, X.; Li, Z. Azobenzene Modified Imidacloprid Derivatives as Photoswitchable Insecticides: Steering Molecular Activity in a Controllable Manner. Sci. Rep. 2015, 5, 1– 8, DOI: 10.1038/srep13962Google ScholarThere is no corresponding record for this reference.
- 68Kabsch, W. Integration, Scaling, Space-Group Assignment and Post-Refinement. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66 (2), 133– 144, DOI: 10.1107/S0907444909047374Google Scholar68https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhs1Sisb4%253D&md5=eb1db34f3c959b4921771eb9f57b2159Integration, scaling, space-group assignment and post-refinementKabsch, WolfgangActa Crystallographica, Section D: Biological Crystallography (2010), 66 (2), 133-144CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)Important steps in the processing of rotation data are described that are common to most software packages. These programs differ in the details and in the methods implemented to carry out the tasks. Here, the working principles underlying the data-redn. package XDS are explained, including the new features of automatic detn. of spot size and reflecting range, recognition and assignment of crystal symmetry and a highly efficient algorithm for the detn. of correction/scaling factors.
- 69McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M. D.; Storoni, L. C.; Read, R. J. Phaser Crystallographic Software. J. Appl. Crystallogr. 2007, 40 (4), 658– 674, DOI: 10.1107/S0021889807021206Google Scholar69https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXnslWqsLk%253D&md5=c63b722ae97e0a74e6a5a079d388f09fPhaser crystallographic softwareMcCoy, Airlie J.; Grosse-Kunstleve, Ralf W.; Adams, Paul D.; Winn, Martyn D.; Storoni, Laurent C.; Read, Randy J.Journal of Applied Crystallography (2007), 40 (4), 658-674CODEN: JACGAR; ISSN:0021-8898. (International Union of Crystallography)Phaser is a program for phasing macromol. crystal structures by both mol. replacement and exptl. phasing methods. The novel phasing algorithms implemented in Phaser have been developed using max. likelihood and multivariate statistics. For mol. replacement, the new algorithms have proved to be significantly better than traditional methods in discriminating correct solns. from noise, and for single-wavelength anomalous dispersion exptl. phasing, the new algorithms, which account for correlations between F+ and F-, give better phases (lower mean phase error with respect to the phases given by the refined structure) than those that use mean F and anomalous differences ΔF. One of the design concepts of Phaser was that it be capable of a high degree of automation. To this end, Phaser (written in C++) can be called directly from Python, although it can also be called using traditional CCP4 keyword-style input. Phaser is a platform for future development of improved phasing methods and their release, including source code, to the crystallog. community.
- 70Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Features and Development of Coot. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66 (4), 486– 501, DOI: 10.1107/S0907444910007493Google Scholar70https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXksFKisb8%253D&md5=67262cbfc60004de5ef962d5c043c910Features and development of CootEmsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K.Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (4), 486-501CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)Coot is a mol.-graphics application for model building and validation of biol. macromols. The program displays electron-d. maps and at. models and allows model manipulations such as idealization, real-space refinement, manual rotation/translation, rigid-body fitting, ligand search, solvation, mutations, rotamers and Ramachandran idealization. Furthermore, tools are provided for model validation as well as interfaces to external programs for refinement, validation and graphics. The software is designed to be easy to learn for novice users, which is achieved by ensuring that tools for common tasks are 'discoverable' through familiar user-interface elements (menus and toolbars) or by intuitive behavior (mouse controls). Recent developments have focused on providing tools for expert users, with customisable key bindings, extensions and an extensive scripting interface. The software is under rapid development, but has already achieved very widespread use within the crystallog. community. The current state of the software is presented, with a description of the facilities available and of some of the underlying methods employed.
- 71Afonine, P. V.; Grosse-Kunstleve, R. W.; Echols, N.; Headd, J. J.; Moriarty, N. W.; Mustyakimov, M.; Terwilliger, T. C.; Urzhumtsev, A.; Zwart, P. H.; Adams, P. D. Towards Automated Crystallographic Structure Refinement with Phenix.Refine. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2012, 68 (4), 352– 367, DOI: 10.1107/S0907444912001308Google Scholar71https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xlt1Ggu7c%253D&md5=1cc7da2cea396f1a75646ea709140cf1Towards automated crystallographic structure refinement with phenix.refineAfonine, Pavel V.; Grosse-Kunstleve, Ralf W.; Echols, Nathaniel; Headd, Jeffrey J.; Moriarty, Nigel W.; Mustyakimov, Marat; Terwilliger, Thomas C.; Urzhumtsev, Alexandre; Zwart, Peter H.; Adams, Paul D.Acta Crystallographica, Section D: Biological Crystallography (2012), 68 (4), 352-367CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)Phenix.refine is a program within the PHENIX package that supports crystallog. structure refinement against exptl. data with a wide range of upper resoln. limits using a large repertoire of model parameterizations. It has several automation features and is also highly flexible. Several hundred parameters enable extensive customizations for complex use cases. Multiple user-defined refinement strategies can be applied to specific parts of the model in a single refinement run. An intuitive graphical user interface is available to guide novice users and to assist advanced users in managing refinement projects. X-ray or neutron diffraction data can be used sep. or jointly in refinement. phenix.refine is tightly integrated into the PHENIX suite, where it serves as a crit. component in automated model building, final structure refinement, structure validation and deposition to the wwPDB. This paper presents an overview of the major phenix.refine features, with extensive literature refs. for readers interested in more detailed discussions of the methods.
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References
This article references 71 other publications.
- 1Velema, W. A.; Van Der Berg, J. P.; Hansen, M. J.; Szymanski, W.; Driessen, A. J. M.; Feringa, B. L. Optical Control of Antibacterial Activity. Nat. Chem. 2013, 5 (11), 924– 928, DOI: 10.1038/nchem.17501https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsVerurzN&md5=ab3ff514012f788c49981666d78acccbOptical control of antibacterial activityVelema, Willem A.; van der Berg, Jan Pieter; Hansen, Mickel J.; Szymanski, Wiktor; Driessen, Arnold J. M.; Feringa, Ben L.Nature Chemistry (2013), 5 (11), 924-928CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)Bacterial resistance is a major problem in the modern world, stemming in part from the build-up of antibiotics in the environment. Novel mol. approaches that enable an externally triggered increase in antibiotic activity with high spatiotemporal resoln. and auto-inactivation are highly desirable. Here, the authors report a responsive, broad-spectrum, antibacterial agent that can be temporally activated with light, whereupon it auto-inactivates on the scale of hours. The use of such a smart antibiotic might prevent the build-up of active antimicrobial material in the environment. Reversible optical control over active drug concn. enables us to obtain pharmacodynamic information. Precisely localized control of activity is achieved, allowing the growth of bacteria to be confined to defined patterns, which has potential for the development of treatments that avoid interference with the endogenous microbial population in other parts of the organism.
- 2Borowiak, M.; Nahaboo, W.; Reynders, M.; Nekolla, K.; Jalinot, P.; Hasserodt, J.; Rehberg, M.; Delattre, M.; Zahler, S.; Vollmar, A.; Trauner, D.; Thorn-Seshold, O. Photoswitchable Inhibitors of Microtubule Dynamics Optically Control Mitosis and Cell Death. Cell 2015, 162 (2), 403– 411, DOI: 10.1016/j.cell.2015.06.0492https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtFCkur7M&md5=0fdc2af4eb68494cbf55e6383a4ecd47Photoswitchable Inhibitors of Microtubule Dynamics Optically Control Mitosis and Cell DeathBorowiak, Malgorzata; Nahaboo, Wallis; Reynders, Martin; Nekolla, Katharina; Jalinot, Pierre; Hasserodt, Jens; Rehberg, Markus; Delattre, Marie; Zahler, Stefan; Vollmar, Angelika; Trauner, Dirk; Thorn-Seshold, OliverCell (Cambridge, MA, United States) (2015), 162 (2), 403-411CODEN: CELLB5; ISSN:0092-8674. (Cell Press)Small mols. that interfere with microtubule dynamics, such as Taxol and the Vinca alkaloids, are widely used in cell biol. research and as clin. anticancer drugs. However, their activity cannot be restricted to specific target cells, which also causes severe side effects in chemotherapy. Here, the authors introduce the photostatins, inhibitors that can be switched on and off in vivo by visible light, to optically control microtubule dynamics. Photostatins modulate microtubule dynamics with a subsecond response time and control mitosis in living organisms with single-cell spatial precision. In longer-term applications in cell culture, photostatins are up to 250 times more cytotoxic when switched on with blue light than when kept in the dark. Therefore, photostatins are both valuable tools for cell biol., and are promising as a new class of precision chemotherapeutics whose toxicity may be spatiotemporally constrained using light.
- 3Hoorens, M. W. H.; Szymanski, W. Reversible, Spatial and Temporal Control over Protein Activity Using Light. Trends Biochem. Sci. 2018, 43 (8), 567– 575, DOI: 10.1016/j.tibs.2018.05.0043https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtFSru7vI&md5=6802043365a055b6a166db2f493fb391Reversible, Spatial and Temporal Control over Protein Activity Using LightHoorens, Mark W. H.; Szymanski, WiktorTrends in Biochemical Sciences (2018), 43 (8), 567-575CODEN: TBSCDB; ISSN:0968-0004. (Elsevier Ltd.)A review. In biomedical sciences, the function of a protein of interest is investigated by altering its net activity and assessing the consequences for the cell or organism. To change the activity of a protein, a wide variety of chem. and genetic tools have been developed. The drawback of most of these tools is that they do not allow for reversible, spatial and temporal control. Here, we describe selected developments in photopharmacol. that aim at establishing such control over protein activity through bioactive mols. with photo-controlled potency. We also discuss why such control is desired and what challenges still need to be overcome for photopharmacol. to reach its maturity as a chem. biol. research tool.
- 4Lerch, M. M.; Hansen, M. J.; van Dam, G. M.; Szymanski, W.; Feringa, B. L. Emerging Targets in Photopharmacology. Angew. Chem., Int. Ed. 2016, 55 (37), 10978– 10999, DOI: 10.1002/anie.2016019314https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtFSqurnN&md5=f2939978a74c93e46e89ccbf6ac9dde4Emerging Targets in PhotopharmacologyLerch, Michael M.; Hansen, Mickel J.; van Dam, Gooitzen M.; Szymanski, Wiktor; Feringa, Ben L.Angewandte Chemie, International Edition (2016), 55 (37), 10978-10999CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The field of photopharmacol. uses mol. photoswitches to establish control over the action of bioactive mols. It aims to reduce systemic drug toxicity and the emergence of resistance, while achieving unprecedented precision in treatment. By using small mols., photopharmacol. provides a viable alternative to optogenetics. We present here a crit. overview of the different pharmacol. targets in various organs and a survey of organ systems in the human body that can be addressed in a non-invasive manner. We discuss the prospects for the selective delivery of light to these organs and the specific requirements for light-activatable drugs. We also aim to illustrate the druggability of medicinal targets with recent findings and emphasize where conceptually new approaches have to be explored to provide photopharmacol. with future opportunities to bring "smart" mol. design ultimately to the realm of clin. use.
- 5Tochitsky, I.; Kienzler, M. A.; Isacoff, E.; Kramer, R. H. Restoring Vision to the Blind with Chemical Photoswitches. Chem. Rev. 2018, 118 (21), 10748– 10773, DOI: 10.1021/acs.chemrev.7b007235https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtVOhsrfI&md5=a953df81c5a7e02a126aad3b14ca9e4fRestoring vision to the blind with chemical photoswitchesTochitsky, Ivan; Kienzler, Michael A.; Isacoff, Ehud; Kramer, Richard H.Chemical Reviews (Washington, DC, United States) (2018), 118 (21), 10748-10773CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Degenerative retinal diseases such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD) affect millions of people around the world and lead to irreversible vision loss if left untreated. A no. of therapeutic strategies have been developed over the years to treat these diseases or restore vision to already blind patients. In this Review, we describe the development and translational application of light-sensitive chem. photoswitches to restore visual function to the blind retina and compare the translational potential of photoswitches with other vision-restoring therapies. This therapeutic strategy is enabled by an efficient fusion of chem. synthesis, chem. biol., and mol. biol. and is broadly applicable to other biol. systems. We hope this Review will be of interest to chemists as well as neuroscientists and clinicians.
- 6Hüll, K.; Morstein, J.; Trauner, D. In Vivo Photopharmacology. Chem. Rev. 2018, 118 (21), 10710– 10747, DOI: 10.1021/acs.chemrev.8b000376https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXht1OmsrrK&md5=9d82ae8b341d68f2e88ec4f6bf3915e0In Vivo PhotopharmacologyHull, Katharina; Morstein, Johannes; Trauner, DirkChemical Reviews (Washington, DC, United States) (2018), 118 (21), 10710-10747CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Synthetic photoswitches have been known for many years, but their usefulness in biol., pharmacol., and medicine has only recently been systematically explored. Over the past decade photopharmacol. has grown into a vibrant field. As the photophys., pharmacodynamic, and pharmacokinetic properties of photoswitches, such as azobenzenes, have become established, they have been applied to a wide range of biol. targets. These include transmembrane proteins (ion channels, transporters, G protein-coupled receptors, receptor-linked enzymes), sol. proteins (kinases, proteases, factors involved in epigenetic regulation), lipid membranes, and nucleic acids. In this review, the authors provide an overview of photopharmacol. using synthetic switches that have been applied in vivo, i.e., in living cells and organisms. The authors discuss the scope and limitations of this approach to study biol. function and the challenges it faces in translational medicine. The relationships between synthetic photoswitches, natural chromophores used in optogenetics, and caged ligands are addressed.
- 7Lundstrom, K. Structural Genomics and Drug Discovery: Molecular Pharmacology. J. Cell. Mol. Med. 2007, 11 (2), 224– 238, DOI: 10.1111/j.1582-4934.2007.00028.x7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXmsFykurw%253D&md5=b2f77376a2586099f6f473d183a9403eStructural genomics and drug discoveryLundstrom, K.Journal of Cellular and Molecular Medicine (2007), 11 (2), 224-238CODEN: JCMMC9; ISSN:1582-1838. (Blackwell Publishing Ltd.)A review. Structure detn. has already proven useful for lead optimization and direct drug design. The no. of high-resoln. structures available in public databases today exceeds 30,000 and will definitely aid in structure-based drug design. Structural genomics approaches covering whole genomes, topol. similar proteins or gene families are great assets for further progress in the development of new drugs. However, membrane proteins representing 70% of current drug targets are poorly characterized structurally. The problems have been related to difficulties in obtaining large amt. of recombinant membrane proteins as well as their purifn. and structure detn. Structural genomics has proven successful in developing new methods in areas from expression to structure detn. by studying a large no. of target proteins in parallel.
- 8Gomez-Santacana, X.; de Munnik, S. M; Mocking, T. A M; Hauwert, N. J; Sun, S.; Vijayachandran, P.; de Esch, I. J P; Vischer, H. F; Wijtmans, M.; Leurs, R. A Toolbox of Molecular Photoswitches to Modulate the CXCR3 Chemokine Receptor with Light. Beilstein J. Org. Chem. 2019, 15 (1), 2509– 2523, DOI: 10.3762/bjoc.15.2448https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitlagtbnE&md5=36f7e7dcf3dd60ddc10a2c3349a3a79aA toolbox of molecular photoswitches to modulate the CXCR3 chemokine receptor with lightGomez-Santacana, Xavier; de Munnik, Sabrina M.; Mocking, Tamara A. M.; Hauwert, Niels J.; Sun, Shanliang; Vijayachandran, Prashanna; de Esch, Iwan J. P.; Vischer, Henry F.; Wijtmans, Maikel; Leurs, RobBeilstein Journal of Organic Chemistry (2019), 15 (), 2509-2523CODEN: BJOCBH; ISSN:1860-5397. (Beilstein-Institut zur Foerderung der Chemischen Wissenschaften)A detailed structure-activity relationship for the scaffold of VUF16216, previously communicated as a small-mol. efficacy photoswitch for the peptidergic chemokine GPCR CXCR3 was reported. A series of photoswitchable azobenzene ligands was prepd. through various synthetic strategies and multistep syntheses. Photochem. and pharmacol. properties were used to guide the design iterations. Investigations of positional and substituent effects reveal that halogen substituents on the ortho-position of the outer ring are preferred for conferring partial agonism on the cis form of the ligands. This effect could be expanded by an electron-donating group on the para-position of the central ring. A variety of efficacy differences between the trans and cis forms emerges from these compds. Tool compds. VUF15888 and VUF16620 represent more subtle efficacy switches, while VUF16216 displays the largest efficacy switch, from antagonism to full agonism. The compd. class disclosed here can aid in new photopharmacol. studies of CXCR3 signaling.
- 9Ricart-Ortega, M.; Font, J.; Llebaria, A. GPCR Photopharmacology. Mol. Cell. Endocrinol. 2019, 488, 36– 51, DOI: 10.1016/j.mce.2019.03.0039https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXltVSgu7g%253D&md5=b11852d80aee99387d1a01a44ab29d61GPCR photopharmacologyRicart-Ortega, Maria; Font, Joan; Llebaria, AmadeuMolecular and Cellular Endocrinology (2019), 488 (), 36-51CODEN: MCEND6; ISSN:0303-7207. (Elsevier Ireland Ltd.)New technologies for spatial and temporal remote control of G protein-coupled receptors (GPCRs) are necessary to unravel the complexity of GPCR signalling in cells, tissues and living organisms. An effective approach, recently developed, consists on the design of light-operated ligands whereby light-dependent GPCR activity regulation can be achieved. In this context, the use of light provides an advantage as it combines safety, easy delivery, high resoln. and it does not interfere with most cellular processes. In this review we summarize the most relevant successful achievements in GPCR photopharmacol. These recent findings constitute a significant advance in research studies on the mol. dynamics of receptor activation and their physiol. roles in vivo. Moreover, these mols. hold potential toward clin. uses as light-operated drugs, which can overcome some of the problems of conventional pharmacol.
- 10Hauwert, N. J.; Mocking, T. A. M.; Da Costa Pereira, D.; Kooistra, A. J.; Wijnen, L. M.; Vreeker, G. C. M.; Verweij, E. W. E.; De Boer, A. H.; Smit, M. J.; De Graaf, C.; Vischer, H. F.; de Esch, I. J. P.; Wijtmans, M.; Leurs, R. Synthesis and Characterization of a Bidirectional Photoswitchable Antagonist Toolbox for Real-Time GPCR Photopharmacology. J. Am. Chem. Soc. 2018, 140 (12), 4232– 4243, DOI: 10.1021/jacs.7b1142210https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXjt1ymtLY%253D&md5=901f689b2d81105aa479089988551a80Synthesis and Characterization of a Bidirectional Photoswitchable Antagonist Toolbox for Real-Time GPCR PhotopharmacologyHauwert, Niels J.; Mocking, Tamara A. M.; Da Costa Pereira, Daniel; Kooistra, Albert J.; Wijnen, Lisa M.; Vreeker, Gerda C. M.; Verweij, Eleonore W. E.; De Boer, Albertus H.; Smit, Martine J.; De Graaf, Chris; Vischer, Henry F.; de Esch, Iwan J. P.; Wijtmans, Maikel; Leurs, RobJournal of the American Chemical Society (2018), 140 (12), 4232-4243CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Noninvasive methods to modulate G protein-coupled receptors (GPCRs) with temporal and spatial precision are in great demand. Photopharmacol. uses photons to control in situ the biol. properties of photoswitchable small-mol. ligands which bodes well for chem. biol. precision approaches. Integrating the light-switchable configurational properties of an azobenzene into the ligand core, the authors developed a bi-directional antagonist toolbox for an archetypical family A GPCR, the histamine H3 receptor (H3R). From 16 newly synthesized photoswitchable compds., VUF14738 (28) and VUF14862 (33) were selected as they swiftly and reversibly photoisomerize and show over 10-fold increased or decreased H3R binding affinities, resp., upon illumination at 360 nm. Both ligands combine long thermal half-lives with fast and high photochem. trans-/cis conversion, allowing their use in real-time electrophysiol. expts. with oocytes to confirm dynamic photomodulation of H3R activation in repeated second-scale cycles. VUF14738 and VUF14862 are robust and fatigue-resistant photoswitchable GPCR antagonists suitable for spatio-temporal studies of H3R signaling.
- 11Bregestovski, P. D.; Maleeva, G. V. Photopharmacology: A Brief Review Using the Control of Potassium Channels as an Example. Neurosci. Behav. Physiol. 2019, 49 (2), 184– 191, DOI: 10.1007/s11055-019-00713-3There is no corresponding record for this reference.
- 12Sansalone, L.; Zhao, J.; Richers, M. T.; Ellis-Davies, G. C. R. Chemical Tuning of Photoswitchable Azobenzenes: A Photopharmacological Case Study Using Nicotinic Transmission. Beilstein J. Org. Chem. 2019, 15 (1), 2812– 2821, DOI: 10.3762/bjoc.15.27412https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitlOqsrjK&md5=85243ba927d37afd01dc1a6a19516e91Chemical tuning of photoswitchable azobenzenes: a photopharmacological case study using nicotinic transmissionSansalone, Lorenzo; Zhao, Jun; Richers, Matthew T.; Ellis-Davies, Graham C. R.Beilstein Journal of Organic Chemistry (2019), 15 (), 2812-2821CODEN: BJOCBH; ISSN:1860-5397. (Beilstein-Institut zur Foerderung der Chemischen Wissenschaften)We have developed photochromic probes for the nicotinic acetylcholine receptor that exploit the unique chem. properties of the tetrafluoroazobenzene (4FAB) scaffold. UV light switching and rapid thermal relaxation of the metastable cis configuration are the main drawbacks assocd. with std. AB-based switches. We designed our photoprobes to take advantage of the excellent thermodn. stability of the cis-4FAB configuration (thermal half-life > 12 days at 37 °C in physiol. buffer) and cis-trans photostationary states above 84%. Furthermore, the well-sepd. n-π* absorption bands of trans- and cis-4FAB allow facile photoswitching with visible light in two optical channels. A convergent 11-step synthetic approach allowed the installation of a trimethylammonium (TA) head onto the 4FAB scaffold, by means of an alkyl spacer, to afford a free diffusible 4FABTA probe. TAs are known to agonize nicotinic receptors, so 4FABTA was tested on mouse brain slices and enabled reversible receptor activation with cycles of violet and green light. Due to the very long-lived metastable cis configuration, 4FAB in vivo use could be of great promise for long term biol. studies. Further chem. functionalization of this 4FAB probe with a maleimide functionality allowed clean crosslinking with glutathione. However, attempts to conjugate with a cysteine on a genetically modified nicotinic acetylcholine receptor did not afford the expected light-responsive channel. Our data indicate that the 4FAB photoswitch can be derivatized bifunctionally for genetically-targeted photopharmacol. while preserving all the favorable photophys. properties of the parent 4FAB scaffold, however, the tetrafluoro motif can significantly perturb pharmacophore-protein interactions. In contrast, we found that the freely diffusible 4FABTA probe could be pre-set with green light into an OFF state that was biol. inert, irradn. with violet light effectively "uncaged" agonist activity, but in a photoreversible manner. Since the neurotransmitter acetylcholine has fully satd. heteroatom valences, our photoswitchable 4FABTA probe could be useful for physiol. studies of this neurotransmitter.
- 13Podewin, T.; Broichhagen, J.; Frost, C.; Groneberg, D.; Ast, J.; Meyer-Berg, H.; Fine, N. H. F.; Friebe, A.; Zacharias, M.; Hodson, D. J.; Trauner, D.; Hoffmann-Röder, A. Optical Control of a Receptor-Linked Guanylyl Cyclase Using a Photoswitchable Peptidic Hormone. Chem. Sci. 2017, 8 (6), 4644– 4653, DOI: 10.1039/C6SC05044A13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmt1ektbo%253D&md5=5f5c7bde048fbd8c90696c275f0f0fbaOptical control of a receptor-linked guanylyl cyclase using a photoswitchable peptidic hormonePodewin, Tom; Broichhagen, Johannes; Frost, Christina; Groneberg, Dieter; Ast, Julia; Meyer-Berg, Helena; Fine, Nicholas H. F.; Friebe, Andreas; Zacharias, Martin; Hodson, David J.; Trauner, Dirk; Hoffmann-Roeder, AnjaChemical Science (2017), 8 (6), 4644-4653CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)The optical control over biol. function with small photoswitchable mols. has gathered significant attention in the last decade. Herein, we describe the design and synthesis of a small library of photoswitchable peptidomimetics based upon human atrial natriuretic peptide (ANP), in which the photochromic amino acid [3-(3-aminomethyl)phenylazo]phenylacetic acid (AMPP) is incorporated into the peptide backbone. The endogeneous hormone ANP signals via the natriuretic peptide receptor A (NPR-A) through raising intracellular cGMP concns., and is involved in blood pressure regulation and sodium homeostasis, as well as lipid metab. and pancreatic function. The cis- and trans-isomers of one of our peptidomimetics, termed TOP271, exhibit a four-fold difference in NPR-A mediated cGMP synthesis in vitro. Despite this seemingly small difference, TOP271 enables large, optically-induced conformational changes ex vivo and transforms the NPR-A into an endogenous photoswitch. Thus, application of TOP271 allows the reversible generation of cGMP using light and remote control can be afforded over vasoactivity in explanted murine aortic rings, as well as pancreatic beta cell function in islets of Langerhans. This study demonstrates the broad applicability of TOP271 to enzyme-dependent signalling processes, extends the toolbox of photoswitchable mols. to all classes of transmembrane receptors and utilizes photopharmacol. to deduce receptor activation on a mol. level.
- 14Beharry, A. A.; Woolley, G. A. Azobenzene Photoswitches for Biomolecules. Chem. Soc. Rev. 2011, 40 (8), 4422– 4437, DOI: 10.1039/c1cs15023e14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXovVemurg%253D&md5=49407115a7f1bc77edbdba90a79c1e58Azobenzene photoswitches for biomoleculesBeharry, Andrew A.; Woolley, G. AndrewChemical Society Reviews (2011), 40 (8), 4422-4437CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. The photoisomerization of azobenzene has been known for almost 75 years but only recently has this process been widely applied to biol. systems. The central challenge of how to productively couple the isomerization process to a large functional change in a biomol. has been met in a no. of instances and it appears that effective photocontrol of a large variety of biomols. may be possible. This crit. review summarizes key properties of azobenzene that enable its use as a photoswitch in biol. systems and describes strategies for using azobenzene photoswitches to drive functional changes in peptides, proteins, nucleic acids, lipids, and carbohydrates (192 refs.).
- 15Babii, O.; Afonin, S.; Berditsch, M.; Reiβer, S.; Mykhailiuk, P. K.; Kubyshkin, V. S.; Steinbrecher, T.; Ulrich, A. S.; Komarov, I. V. Controlling Biological Activity with Light: Diarylethene-Containing Cyclic Peptidomimetics. Angew. Chem., Int. Ed. 2014, 53 (13), 3392– 3395, DOI: 10.1002/anie.20131001915https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXivVertLk%253D&md5=d2e74d71fbe9092e9a531545b4905e28Controlling Biological Activity with Light: Diarylethene-Containing Cyclic PeptidomimeticsBabii, Oleg; Afonin, Sergii; Berditsch, Marina; Reisser, Sabine; Mykhailiuk, Pavel K.; Kubyshkin, Vladimir S.; Steinbrecher, Thomas; Ulrich, Anne S.; Komarov, Igor V.Angewandte Chemie, International Edition (2014), 53 (13), 3392-3395CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Photobiol. processes in nature are usually triggered by nonpeptidic chromophores or by modified side chains. A system is presented in which the polypeptide backbone itself can be conformationally switched by light. An amino acid analog was designed and synthesized based on a reversibly photoisomerizable diarylethene scaffold. This analog was incorporated into the cyclic backbone of the antimicrobial peptide gramicidin S at several sites. The biol. activity of the resulting peptidomimetics could then be effectively controlled by UV/visible light within strictly defined spatial and temporal limits.
- 16Klán, P.; Šolomek, T.; Bochet, C. G.; Blanc, A.; Givens, R.; Rubina, M.; Popik, V.; Kostikov, A.; Wirz, J. Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy. Chem. Rev. 2013, 113 (1), 119– 191, DOI: 10.1021/cr300177k16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhvVyiurvL&md5=b8defe84c6c629e44d429f82fa4eb5f0Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and EfficacyKlan, Petr; Solomek, Tomas; Bochet, Christian G.; Blanc, Aurelien; Givens, Richard; Rubina, Marina; Popik, Vladimir; Kostikov, Alexey; Wirz, JakobChemical Reviews (Washington, DC, United States) (2013), 113 (1), 119-191CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Photoremovable (sometimes called photoreleasable, photocleavable or photoactivatable) protecting groups (PPGs) provide spatial and temporal control over the release of various chems. such as bioagents (neurotransmitters and cell-signaling mols.), acids, bases, Ca2+ ions, oxidants, insecticides, phermones, fragrances, etc. The present review covers recent developments in the field, focusing on the scope, limitations, and applications of PPGs, which are used to release org. mols. Simplified basic structures of PPGs discussed in this review are provided.
- 17Slanina, T.; Shrestha, P.; Palao, E.; Kand, D.; Peterson, J. A.; Dutton, A. S.; Rubinstein, N.; Weinstain, R.; Winter, A. H.; Klán, P. In Search of the Perfect Photocage: Structure-Reactivity Relationships in Meso-Methyl BODIPY Photoremovable Protecting Groups. J. Am. Chem. Soc. 2017, 139 (42), 15168– 15175, DOI: 10.1021/jacs.7b0853217https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhs1Gkur%252FL&md5=93b735b32b8eb44c18391d36fdf0cc47In search of the perfect photocage: structure-reactivity relationships in meso-methyl BODIPY photoremovable protecting groupsSlanina, Tomas; Shrestha, Pradeep; Palao, Eduardo; Kand, Dnyaneshwar; Peterson, Julie A.; Dutton, Andrew S.; Rubinstein, Naama; Weinstain, Roy; Winter, Arthur H.; Klan, PetrJournal of the American Chemical Society (2017), 139 (42), 15168-15175CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)A detailed investigation of the photophys. parameters and photochem. reactivity of meso-Me BODIPY photoremovable protecting groups was accomplished through systematic variation of the leaving group (LG) and core substituents as well as substitutions at boron. Efficiencies of the LG release were evaluated using both steady-state and transient absorption spectroscopies as well as computational analyses to identify the optimal structural features. We find that the quantum yields for photorelease with this photocage are highly sensitive to substituent effects. In particular, we find that the quantum yields of photorelease are improved with derivs. with higher intersystem crossing quantum yields, which can be promoted by core heavy atoms. Moreover, release quantum yields are dramatically improved by boron alkylation, whereas alkylation in the meso-Me position has no effect. Better LGs are released considerably more efficiently than poorer LGs. We find that these substituent effects are additive, for example, a 2,6-diiodo-B-dimethyl BODIPY photocage features quantum yields of 28% for the mediocre LG acetate and a 95% quantum yield of release for chloride. The high chem. and quantum yields combined with the outstanding absorption properties of BODIPY dyes lead to photocages with uncaging cross sections over 10 000 M-1 cm-1, values that surpass cross sections of related photocages absorbing visible light. These new photocages, which absorb strongly near the second harmonic of an Nd:YAG laser (532 nm), hold promise for manipulating and interrogating biol. and material systems with the high spatiotemporal control provided by pulsed laser irradn., while avoiding the phototoxicity problems encountered with many UV-absorbing photocages. More generally, the insights gained from this structure-reactivity relationship may aid in the development of new highly efficient photoreactions.
- 18Deiters, A. Principles and Applications of the Photochemical Control of Cellular Processes. ChemBioChem 2010, 11 (1), 47– 53, DOI: 10.1002/cbic.20090052918https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhs1SqtLjM&md5=83e714f7b6f4260287f0a17bcb2cdf06Principles and Applications of the Photochemical Control of Cellular ProcessesDeiters, AlexanderChemBioChem (2010), 11 (1), 47-53CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Biol. processes, particularly gene function, are naturally regulated with high spatiotemporal resoln. in single cells and multicellular organisms. The activity of genes, proteins, and other biol. mols. is precisely controlled in timing and location. This is esp. evident during the complex biol. processes obsd. in the development of an organism. In order to understand and to study these processes and their misregulation in human disease, it is imperative to control them with the same level of spatiotemporal resoln. found in nature. Here, light irradn. represents a unique tool, because it can be easily and precisely controlled in timing, location, and amplitude; thus, light enables the precise activation and deactivation of biol. function.
- 19Schehr, M.; Ianes, C.; Weisner, J.; Heintze, L.; Müller, M. P.; Pichlo, C.; Charl, J.; Brunstein, E.; Ewert, J.; Lehr, M.; Baumann, U.; Rauh, D.; Knippschild, U.; Peifer, C.; Herges, R. 2-Azo-, 2-Diazocine-Thiazols and 2-Azo-Imidazoles as Photoswitchable Kinase Inhibitors: Limitations and Pitfalls of the Photoswitchable Inhibitor Approach. Photochem. Photobiol. Sci. 2019, 18 (6), 1398– 1407, DOI: 10.1039/C9PP00010K19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXlsleltbg%253D&md5=e30895dd8f7470dd07482c461219509a2-Azo-, 2-diazocine-thiazols and 2-azo-imidazoles as photoswitchable kinase inhibitors: limitations and pitfalls of the photoswitchable inhibitor approachSchehr, Miriam; Ianes, Chiara; Weisner, Joern; Heintze, Linda; Mueller, Matthias P.; Pichlo, Christian; Charl, Julia; Brunstein, Elena; Ewert, Julia; Lehr, Marc; Baumann, Ulrich; Rauh, Daniel; Knippschild, Uwe; Peifer, Christian; Herges, RainerPhotochemical & Photobiological Sciences (2019), 18 (6), 1398-1407CODEN: PPSHCB; ISSN:1474-905X. (Royal Society of Chemistry)In photopharmacol., photoswitchable compds. including azobenzene or other diarylazo moieties exhibit bioactivity against a target protein typically in the slender E-configuration, whereas the rather bulky Z-configuration usually is pharmacol. less potent. Herein we report the design, synthesis and photochem./inhibitory characterization of new photoswitchable kinase inhibitors targeting p38α MAPK and CK1δ. A well characterized inhibitor scaffold was used to attach arylazo- and diazocine moieties. When the isolated isomers, or the photostationary state (PSS) of isomers, were tested in commonly used in vitro kinase assays, however, only small differences in activity were obsd. X-ray analyses of ligand-bound p38α MAPK and CK1δ complexes revealed dynamic conformational adaptations of the protein with respect to both isomers. More importantly, irreversible redn. of the azo group to the corresponding hydrazine was obsd. Independent expts. revealed that reducing agents such as DTT (dithiothreitol) and GSH (glutathione) that are typically used for protein stabilization in biol. assays were responsible. Two further sources of error are the concn. dependence of the E-Z-switching efficiency and artifacts due to incomplete exclusion of light during testing. Our findings may also apply to a no. of previously investigated azobenzene-based photoswitchable inhibitors.
- 20Morgan, H. P.; McNae, I. W.; Nowicki, M. W.; Zhong, W.; Michels, P. A. M.; Auld, D. S.; Fothergill-Gilmore, L. A.; Walkinshaw, M. D. The Trypanocidal Drug Suramin and Other Trypan Blue Mimetics Are Inhibitors of Pyruvate Kinases and Bind to the Adenosine Site. J. Biol. Chem. 2011, 286 (36), 31232– 31240, DOI: 10.1074/jbc.M110.21261320https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtFaitLbO&md5=5aec6da4436d8ef23a0048ad1f3e6158The Trypanocidal Drug Suramin and Other Trypan Blue Mimetics Are Inhibitors of Pyruvate Kinases and Bind to the Adenosine SiteMorgan, Hugh P.; McNae, Iain W.; Nowicki, Matthew W.; Zhong, Wenhe; Michels, Paul A. M.; Auld, Douglas S.; Fothergill-Gilmore, Linda A.; Walkinshaw, Malcolm D.Journal of Biological Chemistry (2011), 286 (36), 31232-31240, S31232/1-S31232/12CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Ehrlich's pioneering chemotherapeutic expts. published in 1904 (Ehrlich, P., and Shiga, K. (1904) Berlin Klin. Wochenschrift 20, 329-362) described the efficacy of a series of dye mols. including trypan blue and trypan red to eliminate trypanosome infections in mice. The mol. structures of the dyes provided a starting point for the synthesis of suramin, which was developed and used as a trypanocidal drug in 1916 and is still in clin. use. Despite the biol. importance of these dye-like mols., the mode of action on trypanosomes has remained elusive. Here the authors present crystal structures of suramin and three related dyes in complex with pyruvate kinases from Leishmania mexicana or from Trypanosoma cruzi. The Ph sulfonate groups of all four mols. (suramin, Ponceau S, acid blue 80, and benzothiazole-2,5-disulfonic acid) bind in the position of ADP/ATP at the active sites of the pyruvate kinases (PYKs). The binding positions in the two different trypanosomatid PYKs are nearly identical. The authors show that suramin competitively inhibits PYKs from humans (muscle, tumor, and liver isoenzymes, Ki = 1.1-17 μM), T. cruzi (Ki = 108 μM), and L. mexicana (Ki = 116 μM), all of which have similar active sites. Synergistic effects were obsd. when examg. suramin inhibition in the presence of an allosteric effector mol., whereby IC50 values decreased up to 2-fold for both trypanosomatid and human PYKs. These kinetic and structural analyses provide insight into the promiscuous inhibition obsd. for suramin and into the mode of action of the dye-like mols. used in Ehrlich's original expts.
- 21Dubay, K. H.; Iwan, K.; Osorio-Planes, L.; Geissler, P. L.; Groll, M.; Trauner, D.; Broichhagen, J. A Predictive Approach for the Optical Control of Carbonic Anhydrase II Activity. ACS Chem. Biol. 2018, 13 (3), 793– 800, DOI: 10.1021/acschembio.7b0086221https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtlyqtbs%253D&md5=f26058ffabe322ca927e715b10b113fcA Predictive Approach for the Optical Control of Carbonic Anhydrase II ActivityDuBay, Kateri H.; Iwan, Katharina; Osorio-Planes, Laura; Geissler, Phillip L.; Groll, Michael; Trauner, Dirk; Broichhagen, JohannesACS Chemical Biology (2018), 13 (3), 793-800CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Optogenetics and photopharmacol. are powerful approaches to investigating biochem. systems. While the former is based on genetically encoded photoreceptors that utilize abundant chromophores, the latter relies on synthetic photoswitches that are either freely diffusible or covalently attached to specific bioconjugation sites, which are often native or engineered cysteines. The identification of suitable cysteine sites and appropriate linkers for attachment is in general a lengthy and cumbersome process. Herein, we describe an in silico screening approach designed to propose a small no. of optimal combinations. By applying this computational approach to human carbonic anhydrase II (hCAII) and a set of three photochromic tethered ligands (PTLs), the no. of potential site-ligand combinations was narrowed from over 750 down to 6, which we then evaluated exptl. Two of these six combinations resulted in light-responsive human Carbonic Anhydrases (LihCAs), which were characterized with enzymic activity assays, mass spectrometry, and X-ray crystallog. Our study also provides insights into the reactivity of cysteines toward maleimides and the hydrolytic stability of the adducts obtained.
- 22Reiter, A.; Skerra, A.; Trauner, D.; Schiefner, A. A Photoswitchable Neurotransmitter Analogue Bound to Its Receptor. Biochemistry 2013, 52 (50), 8972– 8974, DOI: 10.1021/bi401440222https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvVGktLzN&md5=1d77774d2941f27d09018139e820b812A Photoswitchable Neurotransmitter Analogue Bound to Its ReceptorReiter, Alwin; Skerra, Arne; Trauner, Dirk; Schiefner, AndreBiochemistry (2013), 52 (50), 8972-8974CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Incorporation of the azobenzene deriv. gluazo, a synthetic photochromic ligand, into a kainate receptor allows for the optical control of neuronal activity. The crystal structure of gluazo bound to a dimeric GluK2 ligand-binding domain reveals one monomer in a closed conformation, occupied by gluazo, and the other in an open conformation, with a bound buffer mol. The glutamate group of gluazo interacts like the natural glutamate ligand, while its trans-azobenzene moiety protrudes into a tunnel. This elongated cavity presumably cannot accommodate a cis-azobenzene, which explains the reversible activation of the receptor upon photoisomerization.
- 23Cunha, M. R.; Bhardwaj, R.; Lindinger, S.; Butorac, C.; Romanin, C.; Hediger, M. A.; Reymond, J. L. Photoswitchable Inhibitor of the Calcium Channel TRPV6. ACS Med. Chem. Lett. 2019, 10 (9), 1341– 1345, DOI: 10.1021/acsmedchemlett.9b0029823https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhsV2jt7jN&md5=df43f1398f3f6acef072e05b539f249bPhotoswitchable Inhibitor of the Calcium Channel TRPV6Cunha, Micael R.; Bhardwaj, Rajesh; Lindinger, Sonja; Butorac, Carmen; Romanin, Christoph; Hediger, Matthias A.; Reymond, Jean-LouisACS Medicinal Chemistry Letters (2019), 10 (9), 1341-1345CODEN: AMCLCT; ISSN:1948-5875. (American Chemical Society)Herein the authors report the first photoswitchable inhibitor of Transient Receptor Potential Vanilloid 6 (TRPV6), a selective calcium channel involved in a no. of diseases and in cancer progression. By surveying analogs of a previously reported TRPV6 inhibitor appended with a phenyl-diazo group, the authors identified a compd. switching between a weak TRPV6 inhibitor in its dark, E-diazo stereoisomer (Z/E = 3:97, IC50 >> 10 μM) and a potent inhibitor as the Z-diazo stereoisomer accessible reversibly by UV irradn. at λ = 365 nm (Z/E = 3:1, IC50 = 1.7±0.4 μM), thereby allowing precise spatiotemporal control of inhibition. This new tool compd. should be useful to deepen the understanding of TRPV6.
- 24Vandenberg, R. J.; Ryan, R. M. Mechanisms of Glutamate Transport. Physiol. Rev. 2013, 93 (4), 1621– 1657, DOI: 10.1152/physrev.00007.201324https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvVShtrrP&md5=b573b96bbaf6f03c835c471cf2aaf7b8Mechanisms of glutamate transportVandenberg, Robert J.; Ryan, Renae M.Physiological Reviews (2013), 93 (4), 1621-1657CODEN: PHREA7; ISSN:0031-9333. (American Physiological Society)A review. L-Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system and plays important roles in a wide variety of brain functions, but it is also a key player in the pathogenesis of many neurol. disorders. The control of glutamate concns. is crit. to the normal functioning of the central nervous system, and in this review we discuss how glutamate transporters regulate glutamate concns. to maintain dynamic signaling mechanisms between neurons. In 2004, the crystal structure of a prokaryotic homolog of the mammalian glutamate transporter family of proteins was crystd. and its structure detd. This has paved the way for a better understanding of the structural basis for glutamate transporter function. In this review we provide a broad perspective of this field of research, but focus primarily on the more recent studies with a particular emphasis on how our understanding of the structure of glutamate transporters has generated new insights.
- 25Stergachis, A. B.; Pujol-Giménez, J.; Gyimesi, G.; Fuster, D.; Albano, G.; Troxler, M.; Picker, J.; Rosenberg, P. A.; Bergin, A.; Peters, J.; El Achkar, C. M.; Harini, C.; Manzi, S.; Rotenberg, A.; Hediger, M. A.; Rodan, L. H. Recurrent SLC1A2 Variants Cause Epilepsy via a Dominant Negative Mechanism. Ann. Neurol. 2019, 85 (6), 921– 926, DOI: 10.1002/ana.2547725https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXpsFSqtrw%253D&md5=b93ff73eae24f869d28d6df954a90f51Recurrent SLC1A2 variants cause epilepsy via a dominant negative mechanismStergachis, Andrew B.; Pujol-Gimenez, Jonai; Gyimesi, Gergely; Fuster, Daniel; Albano, Giusppe; Troxler, Marina; Picker, Jonathan; Rosenberg, Paul A.; Bergin, Ann; Peters, Jurriaan; El Achkar, Christelle Moufawad; Harini, Chellamani; Manzi, Shannon; Rotenberg, Alexander; Hediger, Matthias A.; Rodan, Lance H.Annals of Neurology (2019), 85 (6), 921-926CODEN: ANNED3; ISSN:0364-5134. (John Wiley & Sons, Inc.)SLC1A2 is a trimeric transporter essential for clearing glutamate from neuronal synapses. Recurrent de novo SLC1A2 missense variants cause a severe, early onset developmental and epileptic encephalopathy via an unclear mechanism. We demonstrate that all 3 variants implicated in this condition localize to the trimerization domain of SLC1A2, and that the Leu85Pro variant acts via a dominant neg. mechanism to reduce, but not eliminate, wild-type SLC1A2 protein localization and function. Finally, we demonstrate that treatment of a 20-mo-old SLC1A2-related epilepsy patient with the SLC1A2-modulating agent ceftriaxone did not result in a significant change in daily spasm count.
- 26Zike, I. D.; Chohan, M. O.; Kopelman, J. M.; Krasnow, E. N.; Flicker, D.; Nautiyal, K. M.; Bubser, M.; Kellendonk, C.; Jones, C. K.; Stanwood, G.; Tanaka, K. F.; Moore, H.; Ahmari, S. E.; Veenstra-VanderWeele, J. OCD Candidate Gene SLC1A1/EAAT3 Impacts Basal Ganglia-Mediated Activity and Stereotypic Behavior. Proc. Natl. Acad. Sci. U. S. A. 2017, 114 (22), 5719– 5724, DOI: 10.1073/pnas.170173611426https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXnslKls7k%253D&md5=3e007fbd35b364481472c7ae284a0b82OCD candidate gene SLC1A1/EAAT3 impacts basal ganglia-mediated activity and stereotypic behaviorZike, Isaac D.; Chohan, Muhammad O.; Kopelman, Jared M.; Krasnow, Emily N.; Flicker, Daniel; Nautiyal, Katherine M.; Bubser, Michael; Kellendonk, Christoph; Jones, Carrie K.; Stanwood, Gregg; Tanaka, Kenji Fransis; Moore, Holly; Ahmari, Susanne E.; Veenstra-VanderWeele, JeremyProceedings of the National Academy of Sciences of the United States of America (2017), 114 (22), 5719-5724CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Obsessive-compulsive disorder (OCD) is a chronic, disabling condition with inadequate treatment options that leave most patients with substantial residual symptoms. Structural, neurochem., and behavioral findings point to a significant role for basal ganglia circuits and for the glutamate system in OCD. Genetic linkage and assocn. studies in OCD point to SLC1A1, which encodes the neuronal glutamate/aspartate/cysteine transporter excitatory amino acid transporter 3 (EAAT3)/excitatory amino acid transporter 1 (EAAC1). However, no previous studies have investigated EAAT3 in basal ganglia circuits or in relation to OCD-related behavior. Here, we report a model of Slc1a1 loss based on an excisable STOP cassette that yields successful ablation of EAAT3 expression and function. Using amphetamine as a probe, we found that EAAT3 loss prevents expected increases in (i) locomotor activity, (ii) stereotypy, and (iii) immediate early gene induction in the dorsal striatum following amphetamine administration. Further, Slc1a1-STOP mice showed diminished grooming in an SKF-38393 challenge expt., a pharmacol. model of OCD-like grooming behavior. This reduced grooming is accompanied by reduced dopamine D1 receptor binding in the dorsal striatum of Slc1a1-STOP mice. Slc1a1-STOP mice also exhibit reduced extracellular dopamine concns. in the dorsal striatum both at baseline and following amphetamine challenge. Viral-mediated restoration of Slc1a1/EAAT3 expression in the midbrain but not in the striatum results in partial rescue of amphetamine-induced locomotion and stereotypy in Slc1a1-STOP mice, consistent with an impact of EAAT3 loss on presynaptic dopaminergic function. Collectively, these findings indicate that the most consistently assocd. OCD candidate gene impacts basal ganglia-dependent repetitive behaviors.
- 27Choi, K. D.; Jen, J. C.; Choi, S. Y.; Shin, J. H.; Kim, H. S.; Kim, H. J.; Kim, J. S.; Choi, J. H. Late-Onset Episodic Ataxia Associated with SLC1A3Mutation. J. Hum. Genet. 2017, 62 (3), 443– 446, DOI: 10.1038/jhg.2016.13727https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXjsVSlsL4%253D&md5=094d25d768839e8262bb7ab920f78e71Late-onset episodic ataxia associated with SLC1A3 mutationChoi, Kwang-Dong; Jen, Joanna C.; Choi, Seo Young; Shin, Jin-Hong; Kim, Hyang-Sook; Kim, Hyo-Jung; Kim, Ji-Soo; Choi, Jae-HwanJournal of Human Genetics (2017), 62 (3), 443-446CODEN: JHGEFR; ISSN:1434-5161. (Nature Publishing Group)Episodic ataxia type 6 (EA6) is caused by mutations in SLC1A3 that encodes excitatory amino acid transporter 1 (EAAT1), a glial glutamate transporter. EAAT1 regulates the extent and durations of glutamate-mediated signal by the clearance of glutamate after synaptic release. In addn., EAAT1 also has an anion channel activity that prevents addnl. glutamate release. We identified a missense mutation in SLC1A3 in a family with EA. The proband exhibited typical EA2-like symptoms such as recurrent ataxia, slurred speech with a duration of several hours, interictal nystagmus and response to acetazolamide, but had late-onset age of sixth decade. Whole-exome sequencing detected a heterozygous c.1177G>A mutation in SLC1A3. This mutation predicted a substitution of isoleucine for a highly conserved valine residue in the seventh transmembrane domain of EAAT1. The mutation was not present in 100 controls, a large panel of inhouse genome data and various mutation databases. Most functional prediction scores revealed to be deleterious. Same heterozygous mutation was identified in one clin. affected family member and two asymptomatic members. Our data expand the mutation spectrum of SLC1A3 and the clin. phenotype of EA6.
- 28Heimer, G.; Marek-Yagel, D.; Eyal, E.; Barel, O.; Oz Levi, D.; Hoffmann, C.; Ruzzo, E. K.; Ganelin-Cohen, E.; Lancet, D.; Pras, E.; Rechavi, G.; Nissenkorn, A.; Anikster, Y.; Goldstein, D. B.; Ben Zeev, B. SLC1A4Mutations Cause a Novel Disorder of Intellectual Disability, Progressive Microcephaly, Spasticity and Thin Corpus Callosum. Clin. Genet. 2015, 88 (4), 327– 335, DOI: 10.1111/cge.1263728https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsFGitLbE&md5=6bca222b398c008b206e1898cd397206SLC1A4 mutations cause a novel disorder of intellectual disability, progressive microcephaly, spasticity and thin corpus callosumHeimer, G.; Marek-Yagel, D.; Eyal, E.; Barel, O.; Oz Levi, D.; Hoffmann, C.; Ruzzo, E. K.; Ganelin-Cohen, E.; Lancet, D.; Pras, E.; Rechavi, G.; Nissenkorn, A.; Anikster, Y.; Goldstein, D. B.; Ben Zeev, B.Clinical Genetics (2015), 88 (4), 327-335CODEN: CLGNAY; ISSN:0009-9163. (Wiley-Blackwell)Two unrelated patients, presenting with significant global developmental delay, severe progressive microcephaly, seizures, spasticity and thin corpus callosum (CC) underwent trio whole-exome sequencing. No candidate variant was found in any known genes related to the phenotype. However, crossing the data of the patients illustrated that they both manifested pathogenic variants in the SLC1A4 gene which codes the ASCT1 transporter of serine and other neutral amino acids. The Ashkenazi patient is homozygous for a deleterious missense c.766G>A, p.(E256K) mutation whereas the Ashkenazi-Iraqi patient is compd. heterozygous for this mutation and a nonsense c.945delTT, p.(Leu315Hisfs*42) mutation. Structural prediction demonstrates truncation of significant portion of the protein by the nonsense mutation and speculates functional disruption by the missense mutation. Both mutations are extremely rare in general population databases, however, the missense mutation was found in heterozygous mode in 1:100 Jewish Ashkenazi controls suggesting a higher carrier rate among Ashkenazi Jews. We conclude that SLC1A4 is the disease causing gene of a novel neurol. disorder manifesting with significant intellectual disability, severe postnatal microcephaly, spasticity and thin CC. The role of SLC1A4 in the serine transport from astrocytes to neurons suggests a possible pathomechanism for this disease and implies a potential therapeutic approach.
- 29Van Geldermalsen, M.; Wang, Q.; Nagarajah, R.; Marshall, A. D.; Thoeng, A.; Gao, D.; Ritchie, W.; Feng, Y.; Bailey, C. G.; Deng, N.; Harvey, K.; Beith, J. M.; Selinger, C. I.; O’Toole, S. A.; Rasko, J. E. J.; Holst, J. ASCT2/SLC1A5 Controls Glutamine Uptake and Tumour Growth in Triple-Negative Basal-like Breast Cancer. Oncogene 2016, 35 (24), 3201– 3208, DOI: 10.1038/onc.2015.38129https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhs1eks73J&md5=9f79dc0c47998d477eb1debe4ad3303aASCT2/SLC1A5 controls glutamine uptake and tumor growth in triple-negative basal-like breast cancervan Geldermalsen, M.; Wang, Q.; Nagarajah, R.; Marshall, A. D.; Thoeng, A.; Gao, D.; Ritchie, W.; Feng, Y.; Bailey, C. G.; Deng, N.; Harvey, K.; Beith, J. M.; Selinger, C. I.; O'Toole, S. A.; Rasko, J. E. J.; Holst, J.Oncogene (2016), 35 (24), 3201-3208CODEN: ONCNES; ISSN:0950-9232. (Nature Publishing Group)Alanine, serine, cysteine-preferring transporter 2 (ASCT2; SLC1A5) mediates uptake of glutamine, a conditionally essential amino acid in rapidly proliferating tumor cells. Uptake of glutamine and subsequent glutaminolysis is crit. for activation of the mTORC1 nutrient-sensing pathway, which regulates cell growth and protein translation in cancer cells. This is of particular interest in breast cancer, as glutamine dependence is increased in high-risk breast cancer subtypes. Pharmacol. inhibitors of ASCT2-mediated transport significantly reduced glutamine uptake in human breast cancer cell lines, leading to the suppression of mTORC1 signaling, cell growth and cell cycle progression. Notably, these effects were subtype-dependent, with ASCT2 transport crit. only for triple-neg. (TN) basal-like breast cancer cell growth compared with minimal effects in luminal breast cancer cells. Both stable and inducible shRNA-mediated ASCT2 knockdown confirmed that inhibiting ASCT2 function was sufficient to prevent cellular proliferation and induce rapid cell death in TN basal-like breast cancer cells, but not in luminal cells. Using a bioluminescent orthotopic xenograft mouse model, ASCT2 expression was then shown to be necessary for both successful engraftment and growth of HCC1806 TN breast cancer cells in vivo. Lower tumoral expression of ASCT2 conferred a significant survival advantage in xenografted mice. These responses remained intact in primary breast cancers, where gene expression anal. showed high expression of ASCT2 and glutamine metab.-related genes, including GLUL and GLS, in a cohort of 90 TN breast cancer patients, as well as correlations with the transcriptional regulators, MYC and ATF4. This study provides preclin. evidence for the feasibility of novel therapies exploiting ASCT2 transporter activity in breast cancer, particularly in the high-risk basal-like subgroup of TN breast cancer where there is not only high expression of ASCT2, but also a marked reliance on its activity for sustained cellular proliferation.
- 30Gebhardt, C.; Körner, R.; Heinemann, U. Delayed Anoxic Depolarizations in Hippocampal Neurons of Mice Lacking the Excitatory Amino Acid Carrier 1. J. Cereb. Blood Flow Metab. 2002, 22 (5), 569– 575, DOI: 10.1097/00004647-200205000-0000830https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XktlWgsL8%253D&md5=9d98ccc74790c2caf4569be146f84c61Delayed anoxic depolarizations in hippocampal neurons of mice lacking the excitatory amino acid carrier 1Gebhardt, Christine; Korner, Rafael; Heinemann, UweJournal of Cerebral Blood Flow and Metabolism (2002), 22 (5), 569-575CODEN: JCBMDN; ISSN:0271-678X. (Lippincott Williams & Wilkins)Hypoxia leads to a rapid increase in vesicular release of glutamate. In addn., hypoxic glutamate release might be caused by reversed operation of neuronal glutamate transporters. An increase in extracellular glutamate concn. might be an important factor in generating anoxic depolarizations (AD) and subsequent neuronal damage. To study the AD and the vesicular release in hippocampal slices from CD1 wild-type mice and mice in which the neuronal glutamate transporter excitatory amino acid carrier 1 (EAAC1) had been knocked out, the authors performed recordings of field potentials and patch clamp recordings of CA1 pyramidal cells. Latency to anoxic depolarizations was enhanced in EAAC1-/- mice, whereas the hypoxia-induced increase in miniature excitatory postsynaptic current frequency occurred with similarly short latencies and to a similar extent in control and mutated animals. Addnl. block of glial glutamate uptake with TBOA (DL-threo-β-benzyloxyaspartate), a nontransportable and potent inhibitor, dramatically reduced the latency to onset of AD and abolished the difference between wild-type mice and EAAC1-/- mice. The authors conclude that the neuronal glutamate transporter greatly influences the latency to generation of AD. Because ADs are not prevented in EAAC1-deficient mice, vesicular release mechanisms also seem to be involved. They become prominent when glial glutamate transport is blocked.
- 31Bjørn-Yoshimoto, W.; Underhill, S. M. The Importance of the Excitatory Amino Acid Transporter 3 (EAAT3). Neurochem. Int. 2016, 98, 4– 18, DOI: 10.1016/j.neuint.2016.05.00731https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2s%252Fkt1KisA%253D%253D&md5=27ff5a73f8de4c0550ebe7ee83660fbdThe importance of the excitatory amino acid transporter 3 (EAAT3)Bjorn-Yoshimoto Walden E; Underhill Suzanne MNeurochemistry international (2016), 98 (), 4-18 ISSN:.The neuronal excitatory amino acid transporter 3 (EAAT3) is fairly ubiquitously expressed in the brain, though it does not necessarily maintain the same function everywhere. It is important in maintaining low local concentrations of glutamate, where its predominant post-synaptic localization can buffer nearby glutamate receptors and modulate excitatory neurotransmission and synaptic plasticity. It is also the main neuronal cysteine uptake system acting as the rate-limiting factor for the synthesis of glutathione, a potent antioxidant, in EAAT3 expressing neurons, while on GABAergic neurons, it is important in supplying glutamate as a precursor for GABA synthesis. Several diseases implicate EAAT3, and modulation of this transporter could prove a useful therapeutic approach. Regulation of EAAT3 could be targeted at several points for functional modulation, including the level of transcription, trafficking and direct pharmacological modulation, and indeed, compounds and experimental treatments have been identified that regulate EAAT3 function at different stages, which together with observations of EAAT3 regulation in patients is giving us insight into the endogenous function of this transporter, as well as the consequences of altered function. This review summarizes work done on elucidating the role and regulation of EAAT3.
- 32Rossi, D. J.; Oshima, T.; Attwell, D. Glutamate Release in Severe Brain Ischaemia Is Mainly by Reversed Uptake. Nature 2000, 403 (6767), 316– 321, DOI: 10.1038/3500209032https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD3c7itlantQ%253D%253D&md5=e8d5d2e38a268b4f3540ea8b69b4fa7cGlutamate release in severe brain ischaemia is mainly by reversed uptakeRossi D J; Oshima T; Attwell DNature (2000), 403 (6767), 316-21 ISSN:0028-0836.The release of glutamate during brain anoxia or ischaemia triggers the death of neurons, causing mental or physical handicap. The mechanism of glutamate release is controversial, however. Four release mechanisms have been postulated: vesicular release dependent on external calcium or Ca2+ released from intracellular stores; release through swelling-activated anion channels; an indomethacin-sensitive process in astrocytes; and reversed operation of glutamate transporters. Here we have mimicked severe ischaemia in hippocampal slices and monitored glutamate release as a receptor-gated current in the CA1 pyramidal cells that are killed preferentially in ischaemic hippocampus. Using blockers of the different release mechanisms, we demonstrate that glutamate release is largely by reversed operation of neuronal glutamate transporters, and that it plays a key role in generating the anoxic depolarization that abolishes information processing in the central nervous system a few minutes after the start of ischaemia. A mathematical model incorporating K+ channels, reversible uptake carriers and NMDA (N-methyl-D-aspartate) receptor channels reproduces the main features of the response to ischaemia. Thus, transporter-mediated glutamate homeostasis fails dramatically in ischaemia: instead of removing extracellular glutamate to protect neurons, transporters release glutamate, triggering neuronal death.
- 33Yaster, M.; Guan, X.; Petralia, R. S.; Rothstein, J. D.; Lu, W.; Tao, Y. X. Effect of Inhibition of Spinal Cord Glutamate Transporters on Inflammatory Pain Induced by Formalin and Complete Freund’s Adjuvant. Anesthesiology 2011, 114 (2), 412– 423, DOI: 10.1097/ALN.0b013e318205df5033https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtFCisLg%253D&md5=61f279c6e65d9ae4d7eebda860bd831eEffect of inhibition of spinal cord glutamate transporters on inflammatory pain induced by formalin and complete freund's adjuvantYaster, Myron; Guan, Xiaowei; Petralia, Ronald S.; Rothstein, Jeffery D.; Lu, Wei; Tao, Yuan-XiangAnesthesiology (2011), 114 (2), 412-423CODEN: ANESAV; ISSN:0003-3022. (Lippincott Williams & Wilkins)Background: Spinal cord glutamate transporters clear synaptically released glutamate and maintain normal sensory transmission. However, their ultrastructural localization is unknown. Moreover, whether and how they participate in inflammatory pain has not been carefully studied. Methods: Immunogold labeling with electron microscopy was carried out to characterize synaptic and nonsynaptic localization of glutamate transporters in the superficial dorsal horn. Their expression and uptake activity after formalin- and complete Freund's adjuvant (CFA)-induced inflammation were evaluated by Western blot anal. and glutamate uptake assay. Effects of intrathecal glutamate transporter activator (R)-(-)-5-methyl-1-nicotinoyl-2-pyrazoline and inhibitors (DL-threo-β-benzyloxyaspartate [TBOA], dihydrokainate, and DL-threo-β-hydroxyaspartate), or TBOA plus group III metabotropic glutamate receptor antagonist (RS)-α-methylserine-O-phosphate, on formalin- and CFA-induced inflammatory pain were examd. Results: In the superficial dorsal horn, excitatory amino acid carrier 1 is localized in presynaptic membrane, postsynaptic membrane, and axonal and dendritic membranes at nonsynaptic sites, whereas glutamate transporter-1 and glutamate/aspartate transporter are prominent in glial membranes. Although expression of these three spinal glutamate transporters was not altered 1 h after formalin injection or 6 h after CFA injection, glutamate uptake activity was decreased at these time points. Intrathecal (R)-(-)-5-methyl-1-nicotinoyl-2-pyrazoline had no effect on formalin-induced pain behaviors. In contrast, intrathecal TBOA, dihydrokainate, and DL-threo-β-hydroxyaspartate reduced formalin-evoked pain behaviors in the second phase. Intrathecal TBOA also attenuated CFA-induced thermal hyperalgesia at 6 h after CFA injection. The antinociceptive effects of TBOA were blocked by coadministration of (RS)-α-methylserine-O-phosphate. Conclusion: Our findings suggest that spinal glutamate transporter inhibition relieves inflammatory pain through activation of inhibitory presynaptic group III metabotropic glutamate receptors.
- 34Winter, N.; Kovermann, P.; Fahlke, C. A Point Mutation Associated with Episodic Ataxia 6 Increases Glutamate Transporter Anion Currents. Brain 2012, 135 (11), 3416– 3425, DOI: 10.1093/brain/aws25534https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3s%252FptVamtg%253D%253D&md5=830b3ac27f7171888bd615534c5b7438A point mutation associated with episodic ataxia 6 increases glutamate transporter anion currentsWinter Natalie; Kovermann Peter; Fahlke ChristophBrain : a journal of neurology (2012), 135 (Pt 11), 3416-25 ISSN:.Episodic ataxia is a human genetic disease characterized by paroxysmal cerebellar incoordination. There are several genetically and clinically distinct forms of this disease, and one of them, episodic ataxia type 6, is caused by mutations in the gene encoding a glial glutamate transporter, the excitatory amino acid transporter-1. So far, reduced glutamate uptake by mutant excitatory amino acid transporter-1 has been thought to be the main pathophysiological process in episodic ataxia type 6. However, excitatory amino acid transporter-1 does not only mediate secondary-active glutamate transport, but also functions as an ion channel. Here, we examined the effects of a disease-associated point mutation, P290R, on glutamate transport, anion current as well as on the subcellular distribution of excitatory amino acid transporter-1 using heterologous expression in mammalian cells. P290R reduces the number of excitatory amino acid transporter-1 in the surface membrane and impairs excitatory amino acid transporter-1-mediated glutamate uptake. Cells expressing P290R excitatory amino acid transporter-1 exhibit larger anion currents than wild-type cells in the absence as well as in the presence of external l-glutamate, despite a lower number of mutant transporters in the surface membrane. Noise analysis revealed unaltered unitary current amplitudes, indicating that P290R modifies opening and closing, and not anion permeation through mutant excitatory amino acid transporter-1 anion channels. These findings identify gain-of-function of excitatory amino acid transporter anion conduction as a pathological process in episodic ataxia. Episodic ataxia type 6 represents the first human disease found to be associated with altered function of excitatory amino acid transporter anion channels and illustrates possible physiological and pathophysiological impacts of this functional mode of this class of glutamate transporters.
- 35Yernool, D.; Boudker, O.; Jin, Y.; Gouaux, E. Structure of a Glutamate Transporter Homologue from Pyrococcus Horikoshii. Nature 2004, 431 (7010), 811– 818, DOI: 10.1038/nature0301835https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXotl2ktL4%253D&md5=1818635a8d3cae0ad8c540a912234cb9Structure of a glutamate transporter homologue from Pyrococcus horikoshiiYernool, Dinesh; Boudker, Olga; Jin, Yan; Gouaux, EricNature (London, United Kingdom) (2004), 431 (7010), 811-818CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Glutamate transporters are integral membrane proteins that catalyze the concentrative uptake of glutamate from the synapse to intracellular spaces by harnessing pre-existing ion gradients. In the central nervous system glutamate transporters are essential for normal development and function, and are implicated in stroke, epilepsy and neurodegenerative diseases. Here we present the crystal structure of a eukaryotic glutamate transporter homolog from Pyrococcus horikoshii. The transporter is a bowl-shaped trimer with a solvent-filled extracellular basin extending halfway across the membrane bilayer. At the bottom of the basin are three independent binding sites, each cradled by two helical hairpins, reaching from opposite sides of the membrane. We propose that transport of glutamate is achieved by movements of the hairpins that allow alternating access to either side of the membrane.
- 36Boudker, O.; Ryan, R. M.; Yernool, D.; Shimamoto, K.; Gouaux, E. Coupling Substrate and Ion Binding to Extracellular Gate of a Sodium-Dependent Aspartate Transporter. Nature 2007, 445 (7126), 387– 393, DOI: 10.1038/nature0545536https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXos12ntQ%253D%253D&md5=f6ad5a2c7b561a8f38bd907db63640d2Coupling substrate and ion binding to extracellular gate of a sodium-dependent aspartate transporterBoudker, Olga; Ryan, Renae M.; Yernool, Dinesh; Shimamoto, Keiko; Gouaux, EricNature (London, United Kingdom) (2007), 445 (7126), 387-393CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Secondary transporters are integral membrane proteins that catalyze the movement of substrate mols. across the lipid bilayer by coupling substrate transport to one or more ion gradients, thereby providing a mechanism for the concentrative uptake of substrates. Here we describe crystallog. and thermodn. studies of GltPh, a sodium (Na+)-coupled aspartate transporter, defining sites for aspartate, two sodium ions and D,L-threo-β-benzyloxyaspartate, an inhibitor. We further show that helical hairpin 2 is the extracellular gate that controls access of substrate and ions to the internal binding sites. At least two sodium ions bind in close proximity to the substrate and these sodium-binding sites, together with the sodium-binding sites in another sodium-coupled transporter, LeuT, define an unwound α-helix as the central element of the ion-binding motif, a motif well suited to the binding of sodium and to participation in conformational changes that accompany ion binding and unbinding during the transport cycle.
- 37Reyes, N.; Ginter, C.; Boudker, O. Transport Mechanism of a Bacterial Homologue of Glutamate Transporters. Nature 2009, 462 (7275), 880– 885, DOI: 10.1038/nature0861637https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhsVentLnE&md5=d663e75e97e7dc7ef66d44b9eb255ea2Transport mechanism of a bacterial homologue of glutamate transportersReyes, Nicolas; Ginter, Christopher; Boudker, OlgaNature (London, United Kingdom) (2009), 462 (7275), 880-885CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Glutamate transporters are integral membrane proteins that catalyze a thermodynamically uphill uptake of the neurotransmitter glutamate from the synaptic cleft into the cytoplasm of glia and neuronal cells by harnessing the energy of pre-existing electrochem. gradients of ions. Crucial to the reaction is the conformational transition of the transporters between outward- and inward-facing states, in which the substrate binding sites are accessible from the extracellular space and the cytoplasm, resp. Here we describe the crystal structure of a double cysteine mutant of a glutamate transporter homolog from Pyrococcus horikoshii, GltPh, which is trapped in the inward-facing state by cysteine crosslinking. Together with the previously detd. crystal structures of GltPh in the outward-facing state, the structure of the crosslinked mutant allows us to propose a mol. mechanism by which GltPh and by analogy, mammalian glutamate transporters, mediate sodium-coupled substrate uptake.
- 38Jensen, S.; Guskov, A.; Rempel, S.; Hänelt, I.; Slotboom, D. J. Crystal Structure of a Substrate-Free Aspartate Transporter. Nat. Struct. Mol. Biol. 2013, 20 (10), 1224– 1226, DOI: 10.1038/nsmb.266338https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtl2ksb%252FN&md5=120e4aa1e318ace8ea037fd946bc6b12Crystal structure of a substrate-free aspartate transporterJensen, Sonja; Guskov, Albert; Rempel, Stephan; Haenelt, Inga; Slotboom, Dirk JanNature Structural & Molecular Biology (2013), 20 (10), 1224-1226CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)Archaeal glutamate transporter homologs catalyze the coupled uptake of aspartate and three sodium ions. After the delivery of the substrate and sodium ions to the cytoplasm, the empty binding site must reorient to the outward-facing conformation to reset the transporter. Here, we report a crystal structure of the substrate-free transporter GltTk from Thermococcus kodakarensis, which provides insight into the mechanism of this essential step in the translocation cycle.
- 39Guskov, A.; Jensen, S.; Faustino, I.; Marrink, S. J.; Slotboom, D. J. Coupled Binding Mechanism of Three Sodium Ions and Aspartate in the Glutamate Transporter Homologue GltTk. Nat. Commun. 2016, 7, 13420, DOI: 10.1038/ncomms1342039https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvVGjtL%252FI&md5=98d108c79c739651594f8d55a68cf304Coupled binding mechanism of three sodium ions and aspartate in the glutamate transporter homologue GltTkGuskov, Albert; Jensen, Sonja; Faustino, Ignacio; Marrink, Siewert J.; Slotboom, Dirk JanNature Communications (2016), 7 (), 13420CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)Glutamate transporters catalyze the thermodynamically unfavorable transport of anionic amino acids across the cell membrane by coupling it to the downhill transport of cations. This coupling mechanism is still poorly understood, in part because the available crystal structures of these transporters are of relatively low resoln. Here we solve crystal structures of the archaeal transporter GltTk in the presence and absence of aspartate and use mol. dynamics simulations and binding assays to show how strict coupling between the binding of three sodium ions and aspartate takes place.
- 40Arkhipova, V.; Guskov, A.; Slotboom, D. J. Structural Ensemble of a Glutamate Transporter Homologue in Lipid Nanodisc Environment. Nat. Commun. 2020, 11, 998, DOI: 10.1038/s41467-020-14834-840https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXkvFahtbY%253D&md5=6b3db8911f70dc8416e76274f2f69872Structural ensemble of a glutamate transporter homologue in lipid nanodisc environmentArkhipova, Valentina; Guskov, Albert; Slotboom, Dirk J.Nature Communications (2020), 11 (1), 998CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Abstr.: Glutamate transporters are cation-coupled secondary active membrane transporters that clear the neurotransmitter L-glutamate from the synaptic cleft. These transporters are homotrimers, with each protomer functioning independently by an elevator-type mechanism, in which a mobile transport domain alternates between inward- and outward-oriented states. Using single-particle cryo-EM we have detd. five structures of the glutamate transporter homolog GltTk, a Na+- L-aspartate symporter, embedded in lipid nanodiscs. Dependent on the substrate concns. used, the protomers of the trimer adopt a variety of asym. conformations, consistent with the independent movement. Six of the 15 resolved protomers are in a hitherto elusive state of the transport cycle in which the inward-facing transporters are loaded with Na+ ions. These structures explain how substrate-leakage is prevented - a strict requirement for coupled transport. The belt protein of the lipid nanodiscs bends around the inward oriented protomers, suggesting that membrane deformations occur during transport.
- 41Alleva, C.; Kovalev, K.; Astashkin, R.; Berndt, M. I.; Baeken, C.; Balandin, T.; Gordeliy, V.; Fahlke, C.; Machtens, J. P. Na+-Dependent Gate Dynamics and Electrostatic Attraction Ensure Substrate Coupling in Glutamate Transporters. Sci. Adv. 2020, 6 (47), eaba9854, DOI: 10.1126/sciadv.aba9854There is no corresponding record for this reference.
- 42Hall, J. L.; Sohail, A.; Cabrita, E. J.; Macdonald, C.; Stockner, T.; Sitte, H. H.; Angulo, J.; MacMillan, F. Saturation Transfer Difference NMR on the Integral Trimeric Membrane Transport Protein GltPh Determines Cooperative Substrate Binding. Sci. Rep. 2020, 10 (1), 1– 9, DOI: 10.1038/s41598-020-73443-zThere is no corresponding record for this reference.
- 43Ruan, Y.; Miyagi, A.; Wang, X.; Chami, M.; Boudker, O.; Scheuring, S. Direct Visualization of Glutamate Transporter Elevator Mechanism by High-Speed AFM. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 1584– 1588, DOI: 10.1073/pnas.161641311443https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslejs74%253D&md5=7b9107bb7a775e962fee210070082a76Direct visualization of glutamate transporter elevator mechanism by high-speed AFMRuan, Yi; Miyagi, Atsushi; Wang, Xiaoyu; Chami, Mohamed; Boudker, Olga; Scheuring, SimonProceedings of the National Academy of Sciences of the United States of America (2017), 114 (7), 1584-1588CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Glutamate transporters are essential for recovery of the neurotransmitter glutamate from the synaptic cleft. Crystal structures in the outward- and inward-facing conformations of a glutamate transporter homolog from archaebacterium Pyrococcus horikoshii, sodium/aspartate symporter GltPh, suggested the mol. basis of the transporter cycle. However, dynamic studies of the transport mechanism have been sparse and indirect. Here, the authors present high-speed at. force microscopy (HS-AFM) observations of membrane-reconstituted GltPh at work. HS-AFM movies provided unprecedented real-space and real-time visualization of the transport dynamics. These results showed transport mediated by large amplitude 1.85-nm "elevator" movements of the transport domains consistent with previous crystallog. and spectroscopic studies. Elevator dynamics occur in the absence and presence of Na+ ions and aspartate, but stall in Na+ alone, providing a direct visualization of the ion and substrate symport mechanism. The authors show unambiguously that individual protomers within the trimeric transporter function fully independently.
- 44Garaeva, A. A.; Slotboom, D. J. Elevator-Type Mechanisms of Membrane Transport. Biochem. Soc. Trans. 2020, 48 (3), 1227– 1241, DOI: 10.1042/BST2020029044https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsV2ku77M&md5=bf1e3c5e303c1ded7450676e30ac1096Elevator-type mechanisms of membrane transportGaraeva, Alisa A.; Slotboom, Dirk J.Biochemical Society Transactions (2020), 48 (3), 1227-1241CODEN: BCSTB5; ISSN:0300-5127. (Portland Press Ltd.)A review. Membrane transporters are integral membrane proteins that mediate the passage of solutes across lipid bilayers. These proteins undergo conformational transitions between outward- and inward-facing states, which lead to alternating access of the substrate-binding site to the aq. environment on either side of the membrane. Dozens of different transporter families have evolved, providing a wide variety of structural solns. to achieve alternating access. A sub-set of structurally diverse transporters operate by mechanisms that are collectively named 'elevator-type'. These transporters have one common characteristic: they contain a distinct protein domain that slides across the membrane as a rigid body, and in doing so it 'drags' the transported substrate along. Anal. of the global conformational changes that take place in membrane transporters using elevator-type mechanisms reveals that elevator-type movements can be achieved in more than one way. Mol. dynamics simulations and exptl. data help to understand how lipid bilayer properties may affect elevator movements and vice versa.
- 45Esslinger, C. S.; Agarwal, S.; Gerdes, J.; Wilson, P. A.; Davis, E. S.; Awes, A. N.; O’Brien, E.; Mavencamp, T.; Koch, H. P.; Poulsen, D. J.; Rhoderick, J. F.; Chamberlin, A. R.; Kavanaugh, M. P.; Bridges, R. J. The Substituted Aspartate Analogue L-β-Threo-Benzyl-Aspartate Preferentially Inhibits the Neuronal Excitatory Amino Acid Transporter EAAT3. Neuropharmacology 2005, 49 (6), 850– 861, DOI: 10.1016/j.neuropharm.2005.08.00945https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtFehsL7I&md5=5bf2793da482cf7bbf27e6a3b0d1bce8The substituted aspartate analogue L-β-threo-benzyl-aspartate preferentially inhibits the neuronal excitatory amino acid transporter EAAT3Esslinger, C. Sean; Agarwal, Shailesh; Gerdes, John; Wilson, Paul A.; Davis, Erin S.; Awes, Alicia N.; O'Brien, Erin; Mavencamp, Teri; Koch, Hans P.; Poulsen, David J.; Rhoderick, Joseph F.; Chamberlin, A. Richard; Kavanaugh, Michael P.; Bridges, Richard J.Neuropharmacology (2005), 49 (6), 850-861CODEN: NEPHBW; ISSN:0028-3908. (Elsevier B.V.)The excitatory amino acid transporters (EAATs) play key roles in the regulation of CNS L-glutamate, esp. related to synthesis, signal termination, synaptic spillover, and excitotoxic protection. Inhibitors available to delineate EAAT pharmacol. and function are essentially limited to those that non-selectively block all EAATs or those that exhibit a substantial preference for EAAT2. Thus, it is difficult to selectively study the other subtypes, particularly EAAT1 and EAAT3. Structure activity studies on a series of β-substituted aspartate analogs identify L-β-benzyl-aspartate (L-β-BA) as among the first blockers that potently and preferentially inhibits the neuronal EAAT3 subtype. Kinetic anal. of D-[3H]aspartate uptake into C17.2 cells expressing the hEAATs demonstrate that L-β-threo-BA is the more potent diastereomer, acts competitively, and exhibits a 10-fold preference for EAAT3 compared to EAAT1 and EAAT2. Electrophysiol. recordings of EAAT-mediated currents in Xenopus oocytes identify L-β-BA as a non-substrate inhibitor. Analyzing L-β-threo-BA within the context of a novel EAAT2 pharmacophore model suggests: a highly conserved positioning of the electrostatic carboxyl and amino groups; nearby regions that accommodate select structural modifications (cyclopropyl rings, Me groups, oxygen atoms); and a unique region L-β-threo-BA occupied by the benzyl moieties of L-TBOA, L-β-threo-BA and related analogs. It is plausible that the preference of L-β-threo-BA and L-TBOA for EAAT3 and EAAT2, resp., could reside in the latter two pharmacophore regions.
- 46Greenfield, A.; Grosanu, C.; Dunlop, J.; McIlvain, B.; Carrick, T.; Jow, B.; Lu, Q.; Kowal, D.; Williams, J.; Butera, J. Synthesis and Biological Activities of Aryl-Ether-, Biaryl-, and Fluorene-Aspartic Acid and Diaminopropionic Acid Analogs as Potent Inhibitors of the High-Affinity Glutamate Transporter EAAT2. Bioorg. Med. Chem. Lett. 2005, 15, 4985– 4988, DOI: 10.1016/j.bmcl.2005.08.00346https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtVynu7zM&md5=a3eab05582346ee6fb135d8134882d6fSynthesis and biological activities of aryl-ether-, biaryl-, and fluorene-aspartic acid and diaminopropionic acid analogs as potent inhibitors of the high-affinity glutamate transporter EAAT-2Greenfield, Alexander; Grosanu, Cristina; Dunlop, John; McIlvain, Beal; Carrick, Tikva; Jow, Brian; Lu, Qiang; Kowal, Dianne; Williams, John; Butera, JohnBioorganic & Medicinal Chemistry Letters (2005), 15 (22), 4985-4988CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)Excitatory amino acid transporters (EAATs) play a pivotal role in maintaining glutamate homeostasis in the mammalian central nervous system, with the EAAT-2 subtype thought to be responsible for the bulk of the glutamate uptake in forebrain regions. A complete elucidation of the functional role of EAAT-2 has been hampered by the lack of potent and selective pharmacol. tools. In this study, the authors describe the synthesis and biol. activities of novel aryl-ether, biaryl-, and fluorene-aspartic acid and diaminopropionic acid analogs as potent inhibitors of EAAT-2. Aspartate deriv. I represents one of the most potent (IC50 = 85 ± 5 nM) and selective inhibitors of EAAT-2 identified to date.
- 47Jensen, A. A.; Erichsen, M. N.; Nielsen, C. W.; Stensbøl, T. B.; Kehler, J.; Bunch, L. Discovery of the First Selective Inhibitor of Excitatory Amino Acid Transporter Subtype 1. J. Med. Chem. 2009, 52 (4), 912– 915, DOI: 10.1021/jm801345847https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtVSqs78%253D&md5=7036a6fe617a9908a36fda6a87400e2cDiscovery of the First Selective Inhibitor of Excitatory Amino Acid Transporter Subtype 1Jensen, Anders A.; Erichsen, Mette N.; Nielsen, Christina W.; Stensboel, Tine B.; Kehler, Jan; Bunch, LennartJournal of Medicinal Chemistry (2009), 52 (4), 912-915CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)The discovery of the first class of subtype-selective inhibitors of the human excitatory amino acid transporter subtype 1 (EAAT1) and its rat orthologue GLAST is reported. An opening structure-activity relationship of 25 analogs is presented that addresses the influence of substitutions at the 4- and 7-positions of the parental skeleton 2-amino-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile. The most potent analog 10 displays high nanomolar inhibitory activity at EAAT1 and a >400-fold selectivity over EAAT2 and EAAT3, making it a highly valuable pharmacol. tool.
- 48Hansen, S. W.; Erichsen, M. N.; Fu, B.; Bjørn-Yoshimoto, W. E.; Abrahamsen, B.; Hansen, J. C.; Jensen, A. A.; Bunch, L. Identification of a New Class of Selective Excitatory Amino Acid Transporter Subtype 1 (EAAT1) Inhibitors Followed by a Structure-Activity Relationship Study. J. Med. Chem. 2016, 59 (19), 8757– 8770, DOI: 10.1021/acs.jmedchem.6b0105848https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsFSjurnK&md5=2151faee0204fe3faff7b003950628dcIdentification of a New Class of Selective Excitatory Amino Acid Transporter Subtype 1 (EAAT1) Inhibitors Followed by a Structure-Activity Relationship StudyHansen, Stinne W.; Erichsen, Mette N.; Fu, Bingru; Bjoern-Yoshimoto, Walden E.; Abrahamsen, Bjarke; Hansen, Jacob C.; Jensen, Anders A.; Bunch, LennartJournal of Medicinal Chemistry (2016), 59 (19), 8757-8770CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Screening of a small compd. library at three excitatory amino acid transporter subtypes 1-3 (EAAT1-3) subtypes resulted in the identification of compd. (I) that exhibited a distinct preference as an inhibitor at EAAT1 (IC50 20 μM) compared to EAAT2 and EAAT3 (IC50 > 300 μM). This prompted us to subject I to an elaborate structure-activity-relationship study through the purchase and synthesis and subsequent pharmacol. characterization of a total of 36 analogs. Although this effort did not result in analogs with substantially improved inhibitory potencies at EAAT1 compared to that displayed by the hit, it provided a detailed insight into structural requirements for EAAT1 activity of this scaffold. The discovery of this new class of EAAT1-selective inhibitors not only supplements the currently available pharmacol. tools in the EAAT field, but also substantiates the notion that EAAT ligands not derived from α-amino acids hold considerable potential in terms of subtype-selective modulation.
- 49Abrahamsen, B.; Schneider, N.; Erichsen, M. N.; Huynh, T. H. V.; Fahlke, C.; Bunch, L.; Jensen, A. A. Allosteric Modulation of an Excitatory Amino Acid Transporter: The Subtype-Selective Inhibitor UCPH-101 Exerts Sustained Inhibition of EAAT1 through an Intramonomeric Site in the Trimerization Domain. J. Neurosci. 2013, 33 (3), 1068– 1087, DOI: 10.1523/JNEUROSCI.3396-12.201349https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsVemsLg%253D&md5=5e2b79367256a04008e6180cb9856d6fAllosteric modulation of an excitatory amino acid transporter:the subtype-selective inhibitor UCPH-101 exerts sustained inhibition of EAAT1 through an intramonomeric site in the trimerization domainAbrahamsen, Bjarke; Schneider, Nicole; Erichsen, Mette N.; Huynh, Tri H. V.; Fahlke, Christoph; Bunch, Lennart; Jensen, Anders A.Journal of Neuroscience (2013), 33 (3), 1068-1087CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)In the present study, the mechanism of action and mol. basis for the activity of the first class of selective inhibitors of the human excitatory amino acid transporter subtype 1 (EAAT1) and its rodent ortholog GLAST are elucidated. The previously reported specificity of UCPH-101 and UCPH-102 for EAAT1 over EAAT2 and EAAT3 is demonstrated to extend to the EAAT4 and EAAT5 subtypes as well. Interestingly, brief exposure to UCPH-101 induces a long-lasting inactive state of EAAT1, whereas the inhibition exerted by closely related analogs is substantially more reversible in nature. In agreement with this, the kinetic properties of UCPH-101 unblocking of the transporter are considerably slower than those of UCPH-102. UCPH-101 exhibits noncompetitive inhibition of EAAT1, and its binding site in GLAST has been delineated in an elaborate mutagenesis study. Substitutions of several residues in TM3, TM4c, and TM7a of GLAST have detrimental effects on the inhibitory potency and/or efficacy of UCPH-101 while not affecting the pharmacol. properties of (S)-glutamate or the competitive EAAT inhibitor TBOA significantly. Hence, UCPH-101 is proposed to target a predominantly hydrophobic crevice in the "trimerization domain" of the GLAST monomer, and the inhibitor is demonstrated to inhibit the uptake through the monomer that it binds to exclusively and not to affect substrate translocation through the other monomers in the GLAST trimer. The allosteric mode of UCPH-101 inhibition underlines the functional importance of the trimerization domain of the EAAT and demonstrates the feasibility of modulating transporter function through ligand binding to regions distant from its "transport domain.".
- 50Balcar, V. J.; Johnston, G. A. R. The Structural Specificity of the High Affinity Uptake of L-Glutamate and L-Aspartate by Rat Brain Slices. J. Neurochem. 1972, 19 (11), 2657– 2666, DOI: 10.1111/j.1471-4159.1972.tb01325.x50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE3sXmslKhtw%253D%253D&md5=3a14f9449d9764ca8f1ee9f48d8532b4Structural specificity of the high-affinity uptake of L-glutamate and L-aspartate by rat brain slicesBalcar, V. J.; Johnston, G. A. R.Journal of Neurochemistry (1972), 19 (11), 2657-66CODEN: JONRA9; ISSN:0022-3042.The high-affinity uptake system for L-glutamate [56-86-0] and L-aspartate [56-84-8] in rat cerebral cortex may not be specific for these likely excitatory synaptic transmitters, since threo-3-hydroxy-DL-aspartate [4294-45-5], L-cysteinesulfinate [15932-83-9], L-cysteate [498-40-8], and D-aspartate [1783-96-6] strongly inhibited the obsd. high-affinity uptake of L-glutamate by rat brain slices in a manner consistent with linear competitive inhibition. L-glutamate high-affinity uptake was Na+-dependent, but under certain conditions appeared to be less sensitive than γ-aminobutyrate uptake to changes in the external Na+ concn. and to drugs which modify Na+ movements. This may be relevant to the efficiency of the glutamate uptake process during synaptic depolarization induced by glutamate. L-glutamate high-affinity uptake was also inhibited in a relatively nonspecific manner by a variety of drugs including mercurials and some electron transport inhibitors.
- 51Shimamoto, K.; LeBrun, B.; Yasuda-Kamatani, Y.; Sakaitani, M.; Shigeri, Y.; Yumoto, N.; Nakajima, T. DL-Threo-Beta-Benzyloxyaspartate, a Potent Blocker of Excitatory Amino Acid Transporters. Mol. Pharmacol. 1998, 53, 195– 201, DOI: 10.1124/mol.53.2.19551https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXht12htrw%253D&md5=90eb235209edbbcaa9bf5a7e69dae743DL-threo-β-Benzyloxyaspartate, a potent blocker of excitatory amino acid transportersShimamoto, Keiko; Lebrun, Bruno; Yasuda-Kamatani, Yoshimi; Sakaitani, Masahiro; Shigeri, Yasushi; Yumoto, Noboru; Nakajima, TerumiMolecular Pharmacology (1998), 53 (2), 195-201CODEN: MOPMA3; ISSN:0026-895X. (Williams & Wilkins)DL-Threo-β-Benzyloxyaspartate (DL-TBOA), a novel deriv. of DL-threo-β-hydroxyaspartate, was synthesized and examd. as an inhibitor of sodium-dependent glutamate/aspartate (excitatory amino acid) transporters. DL-TBOA inhibited the uptake of [14C]glutamate in COS-1 cells expressing the human excitatory amino acid transporter-1 (EAAT1) (Ki = 42 μM) with almost the same potency as DL-threo-β-hydroxyaspartate (Ki = 58 μM). With regard to the human excitatory amino acid transporter-2 (EAAT2), the inhibitory effect of DL-TBOA (Ki = 5.7 μM) was much more potent than that of dihydrokainate (Ki = 79 μM), which is well known as a selective blocker of this subtype. Electrophysiol., DL-TBOA induced no detectable inward currents in Xenopus laevis oocytes expressing human EAAT1 or EAAT2. However, it significantly reduced the glutamate-induced currents, indicating the prevention of transport. The dose-response curve of glutamate was shifted by adding DL-TBOA without a significant change in the max. current. The Kb values for human EAAT1 and EAAT2 expressed in X. laevis oocytes were 9.0 μM and 116 nM, resp. These results demonstrated that DL-TBOA is, so far, the most potent competitive blocker of glutamate transporters. DL-TBOA did not show any significant effects on either the ionotropic or metabotropic glutamate receptors. Moreover, DL-TBOA is chem. much more stable than its benzoyl analog, a previously reported blocker of excitatory amino acid transporters; therefore, DL-TBOA should be a useful tool for investigating the physiol. roles of transporters.
- 52Shimamoto, K.; Sakai, R.; Takaoka, K.; Yumoto, N.; Nakajima, T.; Amara, S. G.; Shigeri, Y. Characterization of Novel L-Threo-Beta-Benzyloxyaspartate Derivatives, Potent Blockers of the Glutamate Transporters. Mol. Pharmacol. 2004, 65 (4), 1008– 1015, DOI: 10.1124/mol.65.4.100852https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXivFaltbo%253D&md5=48cc0d672b697001a4161172920406e8Characterization of novel L-threo-β-benzyloxyaspartate derivatives, potent blockers of the glutamate transportersShimamoto, Keiko; Sakai, Ryuichi; Takaoka, Kiyo; Yumoto, Noboru; Nakajima, Terumi; Amara, Susan G.; Shigeri, YasushiMolecular Pharmacology (2004), 65 (4), 1008-1015CODEN: MOPMA3; ISSN:0026-895X. (American Society for Pharmacology and Experimental Therapeutics)Nontransportable blockers of the glutamate transporters are important tools for investigating mechanisms of synaptic transmission. DL-threo-β-Benzyloxyaspartate (DL-TBOA) is a potent blocker of all subtypes of the excitatory amino acid transporters (EAATs). We characterized novel L-TBOA analogs possessing a substituent on their resp. benzene rings. The analogs significantly inhibited labeled glutamate uptake, the most potent of which was (2S,3S)-3-{3-[4-(trifluoromethyl)benzoylamino]benzyloxy}aspartate (TFB-TBOA). In an uptake assay using cells transiently expressing EAATs, the IC50 values of TFB-TBOA for EAAT1, EAAT2, and EAAT3 were 22, 17, and 300 nM, resp. TFB-TBOA was significantly more potent at inhibiting EAAT1 and EAAT2 compared with L-TBOA (IC50 values for EAAT1-3 were 33, 6.2, and 15 μM, resp.). Electrophysiol. analyses revealed that TBOA analogs block the transport-assocd. currents in all five EAAT subtypes and also block leak currents in EAAT5. The rank order of the analogs for potencies at inhibiting substrate-induced currents was identical to that obsd. in the uptake assay. However, the kinetics of TFB-TBOA differed from the kinetics of L-TBOA, probably because of the strong binding affinity. Notably, TFB-TBOA did not affect other representative neurotransmitter transporters or receptors, including ionotropic and metabotropic glutamate receptors, indicating that it is highly selective for EAATs. Moreover, intracerebroventricular administration of the TBOA analogs induced severe convulsive behaviors in mice, probably because of the accumulation of glutamate. Taken together, these findings indicate that novel TBOA analogs, esp. TFB-TBOA, should serve as useful tools for elucidating the physiol. roles of the glutamate transporters.
- 53Fu, H.; Zhang, J.; Tepper, P. G.; Bunch, L.; Jensen, A. A.; Poelarends, G. J. Chemoenzymatic Synthesis and Pharmacological Characterization of Functionalized Aspartate Analogues as Novel Excitatory Amino Acid Transporter Inhibitors. J. Med. Chem. 2018, 61 (17), 7741– 7753, DOI: 10.1021/acs.jmedchem.8b0070053https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtlantb3E&md5=7cc9859a2743c381856009492c23de2cChemoenzymatic Synthesis and Pharmacological Characterization of Functionalized Aspartate Analogues As Novel Excitatory Amino Acid Transporter InhibitorsFu, Haigen; Zhang, Jielin; Tepper, Pieter G.; Bunch, Lennart; Jensen, Anders A.; Poelarends, Gerrit J.Journal of Medicinal Chemistry (2018), 61 (17), 7741-7753CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)Aspartate (Asp) derivs. are privileged compds. for investigating the roles governed by excitatory amino acid transporters (EAATs) in glutamatergic neurotransmission. Here, we report the synthesis of various Asp derivs. with (cyclo)alkyloxy and (hetero)aryloxy substituents at C-3. Their pharmacol. properties were characterized at the EAAT1-4 subtypes. The L-threo-3-substituted Asp derivs. (I) (R1 = cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, 2-thiophenyl, 3-thiophenyl, 2-furanyl, 3-furanyl, propargyl) were non-substrate inhibitors, exhibiting pan activity at EAAT1-4 with IC50 values ranging from 0.49 to 15 μM. Comparisons between (DL-threo)- and (DL-erythro)-Asp analogs I (R1 = cyclopropyl, 3-thiophenyl, propargyl) confirmed that the threo configuration is crucial for the EAAT1-4 inhibitory activities. Analogs I (R1 = cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl) of L-TFB-TBOA I (R1 = cyclopropyl) were shown to be potent EAAT1-4 inhibitors, with IC50 values ranging from 5-530 nM. Hybridization of the nonselective EAAT inhibitor L-TBOA with EAAT2-selective inhibitor WAY-213613 or EAAT3-preferring inhibitor NBI-59159 yielded compds. (II)(R2 = III and IV) resp., which were non-selective EAAT inhibitors displaying considerably lower IC50 values at EAAT1-4 (11-140 nM) than those displayed by the resp. parent mols.
- 54Canul-Tec, J. C.; Assal, R.; Cirri, E.; Legrand, P.; Brier, S.; Chamot-Rooke, J.; Reyes, N. Structure and Allosteric Inhibition of Excitatory Amino Acid Transporter 1. Nature 2017, 544, 446– 451, DOI: 10.1038/nature2206454https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmtlalt7k%253D&md5=1107e965f53e44801b6f51152f29ba13Structure and allosteric inhibition of excitatory amino acid transporter 1Canul-Tec, Juan C.; Assal, Reda; Cirri, Erica; Legrand, Pierre; Brier, Sebastien; Chamot-Rooke, Julia; Reyes, NicolasNature (London, United Kingdom) (2017), 544 (7651), 446-451CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Human members of the solute carrier 1 (SLC1) family of transporters take up excitatory neurotransmitters in the brain and amino acids in peripheral organs. Dysregulation of the function of SLC1 transporters is assocd. with neurodegenerative disorders and cancer. Here we present crystal structures of a thermostabilized human SLC1 transporter, the excitatory amino acid transporter 1 (EAAT1), with and without allosteric and competitive inhibitors bound. The structures reveal architectural features of the human transporters, such as intra- and extracellular domains that have potential roles in transport function, regulation by lipids and post-translational modifications. The coordination of the allosteric inhibitor in the structures and the change in the transporter dynamics measured by hydrogen-deuterium exchange mass spectrometry reveal a mechanism of inhibition, in which the transporter is locked in the outward-facing states of the transport cycle. Our results provide insights into the mol. mechanisms underlying the function and pharmacol. of human SLC1 transporters.
- 55Garaeva, A. A.; Guskov, A.; Slotboom, D. J.; Paulino, C. A One-Gate Elevator Mechanism for the Human Neutral Amino Acid Transporter ASCT2. Nat. Commun. 2019, 10 (1), 3427, DOI: 10.1038/s41467-019-11363-x55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3MvjsVensg%253D%253D&md5=bdb8e27fd66ac8d3a59fc28b3740d9d1A one-gate elevator mechanism for the human neutral amino acid transporter ASCT2Garaeva Alisa A; Slotboom Dirk J; Paulino Cristina; Guskov Albert; Paulino Cristina; Slotboom Dirk JNature communications (2019), 10 (1), 3427 ISSN:.The human Alanine Serine Cysteine Transporter 2 (ASCT2) is a neutral amino acid exchanger that belongs to the solute carrier family 1 (SLC1A). SLC1A structures have revealed an elevator-type mechanism, in which the substrate is translocated across the cell membrane by a large displacement of the transport domain, whereas a small movement of hairpin 2 (HP2) gates the extracellular access to the substrate-binding site. However, it has remained unclear how substrate binding and release is gated on the cytoplasmic side. Here, we present an inward-open structure of the human ASCT2, revealing a hitherto elusive SLC1A conformation. Strikingly, the same structural element (HP2) serves as a gate in the inward-facing as in the outward-facing state. The structures reveal that SLC1A transporters work as one-gate elevators. Unassigned densities near the gate and surrounding the scaffold domain, may represent potential allosteric binding sites, which could guide the design of lipidic-inhibitors for anticancer therapy.
- 56Cheng, B.; Trauner, D.; Shchepakin, D.; Kavanaugh, M. P. A Photoswitchable Inhibitor of a Glutamate Transporter. ACS Chem. Neurosci. 2017, 8, 1668– 1672, DOI: 10.1021/acschemneuro.7b0007256https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmtFCmuro%253D&md5=7a430f138b3da62ba24ff4ceb2f7a681Photoswitchable Inhibitor of a Glutamate TransporterCheng, Bichu; Shchepakin, Denis; Kavanaugh, Michael P.; Trauner, DirkACS Chemical Neuroscience (2017), 8 (8), 1668-1672CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)Excitatory amino acid transporters clear glutamate from the synaptic cleft and play a crit. role in glutamatergic neurotransmission. Their differential roles in astrocytes, microglia, and neurons are poorly understood due in part to a lack of pharmacol. tools that can be targeted to specific cells and tissues. We now describe a photoswitchable inhibitor, termed ATT, that interacts with the major mammalian forebrain transporters EAAT1-3 in a manner that can be reversibly switched between trans (high-affinity) and cis (low-affinity) configurations using light of different colors. In the dark, ATT competitively inhibited the predominant glial transporter EAAT2 with ∼200-fold selectivity over the neuronal transporter EAAT3. Brief exposure to 350 nm light reduced the steady-state blocker affinity by more than an order of magnitude. Illumination of EAAT2 complexed with ATT induced a corresponding increase in the blocker off-rate monitored in the presence of glutamate. ATT can be used to reversibly manipulate glutamate transporter activity with light and may be useful to gain insights into the dynamic physiol. roles of glutamate transporters in the brain, as well as to study the mol. interactions of transporters with ligands.
- 57Hoorens, M. W. H.; Fu, H.; Duurkens, R. H.; Trinco, G.; Arkhipova, V.; Feringa, B. L.; Poelarends, G. J.; Slotboom, D. J.; Szymanski, W. Glutamate Transporter Inhibitors with Photo-Controlled Activity. Adv. Ther. 2018, 1, 1800028, DOI: 10.1002/adtp.201800028There is no corresponding record for this reference.
- 58Foster, A. C.; Li, Y.-X.; Runyan, S.; Dinh, T.; Venadas, S.; Chen, J.; Pashikanti, S.; Datta, A.; Ehring, G.; Staubli, U. Activity of the Enantiomers of Erythro-3-Hydroxyaspartate at Glutamate Transporters and NMDA Receptors. J. Neurochem. 2016, 136, 692– 697, DOI: 10.1111/jnc.1343058https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXitVemtLnE&md5=0e9c224d70b60bf40cab4f0133c5ebbdActivity of the enantiomers of erythro-3-hydroxyaspartate at glutamate transporters and NMDA receptorsFoster, Alan C.; Li, Yong-Xin; Runyan, Stephen; Dinh, Tim; Venadas, Steven; Chen, June; Pashikanti, Srinath; Datta, Apurba; Ehring, George; Staubli, UrsulaJournal of Neurochemistry (2016), 136 (4), 692-697CODEN: JONRA9; ISSN:0022-3042. (Wiley-Blackwell)The enantiomers of erythro-3-hydroxyaspartate were tested for activity at glutamate transporters and NMDA receptors. Both enantiomers inhibited glutamate transporters in rat hippocampal crude synaptosomes and elicited substrate-like activity at excitatory amino acid transporter 1, 2, and 3 as measured by voltage clamp in the Xenopus oocyte expression system. The enantiomers had similar affinities, but the D-enantiomer showed a lower maximal effect at excitatory amino acid transporter 1, 2, and 3 than the L-enantiomer. Surprisingly, D-erythro-3-hydroxyaspartate was a potent NMDA receptor agonist with an EC50 value in rat hippocampal neurons of 320 nM, whereas the L-enantiomer was 100-fold less potent. L-erythro-3-hydroxyaspartate showed activity at both glutamate transporters and NMDA receptors at concns. that are reported to inhibit serine racemase, indicating a lack of selectivity. This enantiomeric pair may assist in shedding further light on the structural requirements for substrate activity at glutamate transporters and for agonist activity at NMDA receptors. The erythro enantiomers of 3-hydroxyaspartate had interesting and surprising effects on glutamate neurotransmitter systems. L-erythro-3-hydroxyaspartate had activity at both glutamate transporters (EAAT1/2/3) and NMDA receptors. D-erythro-3-hydroxyaspartate acted on EAATs, but was also identified as a highly potent NMDA receptor agonist. These enantiomers shed further light on the structural requirements for activity at EAATs and NMDA receptors.
- 59Tolner, B.; Ubbink-Kok, T.; Poolman, B.; Konings, W. N. Characterization of the Proton/Glutamate Symport Protein of Bacillus Subtilis and Its Functional Expression in Escherichia Coli. J. Bacteriol. 1995, 177 (10), 2863– 2869, DOI: 10.1128/JB.177.10.2863-2869.199559https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXls1Sks7w%253D&md5=a96a4474c5a9f3fd1c07837b8dadfb72Characterization of the proton/glutamate symport protein of Bacillus subtilis and its functional expression in Escherichia coliTolner, Berend; Ubbink-Kok, Trees; Poolman, Bert; Konings, Wil N.Journal of Bacteriology (1995), 177 (10), 2863-9CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)Transport of acidic amino acids in Bacillus subtilis is an electrogenic process in which L-glutamate or L-aspartate is symported with at least two protons. This is shown by studies of transport in membrane vesicles in which a proton motive force is generated by oxidn. of ascorbate-phenazine methosulfate or by artificial ion gradients. An inwards-directed sodium gradient had no (stimulatory) effect on proton motive force-driven L-glutamate uptake. The transporter is specific for L-glutamate and L-aspartate. L-Glutamate transport is inhibited by β-hydroxyaspartate and cysteic acid but not by α-methyl-glutamate. The gene encoding the L-glutamate transport protein of B. subtilis (gltPBsu) was cloned by complementation of Escherichia coli JC5412 for growth on glutamate as the sole source of carbon, energy, and nitrogen, and its nucleotide sequence was detd. Putative promoter, terminator, and ribosome binding site sequences were found in the flanking regions. UUG is most likely the start codon. GltPBsu encodes a polypeptide of 414 amino acid residues and is homologous to several proteins that transport glutamate and/or structurally related compds. such as aspartate, fumarate, malate, and succinate. Both sodium- and proton-coupled transporters belong to this family of dicarboxylate transporters. Hydropathy profiling and multiple alignment of the fatty of carboxylate transporters suggest that each of the proteins spans the cytoplasmic membrane 12 times with both the amino and carboxy termini on the inside.
- 60Tolner, B.; Ubbink-Kok, T.; Poolmann, B.; Konings, W. N. Cation-Selectivity of the L-Glutamate Transporters of Escherichia Coli, Bacillus Stearothermophilus and Bacillus Caldotenax: Dependence on the Environment in Which the Proteins Are Expressed. Mol. Microbiol. 1995, 18 (1), 123– 133, DOI: 10.1111/j.1365-2958.1995.mmi_18010123.x60https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXptlersbw%253D&md5=54084c9847f378f805022850afd0122cCation-selectivity of the L-glutamate transporters of Escherichia coli, Bacillus stearothermophilus and Bacillus caldotenax: dependence on the environment in which the proteins are expressedTolner, Berend; Ubbink-Kok, Trees; Poolman, Bert; Konings, Will N.Molecular Microbiology (1995), 18 (1), 123-33CODEN: MOMIEE; ISSN:0950-382X. (Blackwell)L-Glutamate transport by the H+-glutamate and Na+-glutamate symport proteins of Escherichia coli K-12 (GltPEc and GltSEc, resp.) and the Na+-H+-glutamate symport proteins of Bacillus stearothermophilus (GltTBs) and Bacillus caldotenax (GltTBc) was studied in membrane vesicles derived from cells in which the proteins were either homologously or heterologously expressed. Substrate and inhibitor specificity studies indicate that GltPEc, GltTBs and GltTBc fall into the same group of transporters, whereas GltSEc is distinctly different from the others. Also, the cation specificity of GltSEc is different; GltSEc transported L-glutamate with (at least) two Na+, whereas GltPEc, GltTBs and GltTBc catalyzed an electrogenic symport of L-glutamate with ≥two H+, i.e. when the proteins were expressed in E. coli. Surprisingly studies in membrane vesicles of B. stearothermophilus and B. caldotenax indicated a Na+-H+-L-glutamate symport for both GltTBs and GltTBc. The Na+ dependency of the GltT transporters in the Bacillus strains increased with temp. These observations suggest that the conformation of the transport proteins in the E. coli and the Bacillus membranes differs, which influences the coupling ion selectivity.
- 61Woodhouse, J.; Nass Kovacs, G.; Coquelle, N.; Uriarte, L. M.; Adam, V.; Barends, T. R. M.; Byrdin, M.; de la Mora, E.; Bruce Doak, R.; Feliks, M.; Field, M.; Fieschi, F.; Guillon, V.; Jakobs, S.; Joti, Y.; Macheboeuf, P.; Motomura, K.; Nass, K.; Owada, S.; Roome, C. M.; Ruckebusch, C.; Schirò, G.; Shoeman, R. L.; Thepaut, M.; Togashi, T.; Tono, K.; Yabashi, M.; Cammarata, M.; Foucar, L.; Bourgeois, D.; Sliwa, M.; Colletier, J.-P.; Schlichting, I.; Weik, M. Photoswitching Mechanism of a Fluorescent Protein Revealed by Time-Resolved Crystallography and Transient Absorption Spectroscopy. Nat. Commun. 2020, 11, 741, DOI: 10.1038/s41467-020-14537-061https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXjs1OhtLY%253D&md5=1564ff90993aa05bf9fad976808fc853Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopyWoodhouse, Joyce; Nass Kovacs, Gabriela; Coquelle, Nicolas; Uriarte, Lucas M.; Adam, Virgile; Barends, Thomas R. M.; Byrdin, Martin; de la Mora, Eugenio; Bruce Doak, R.; Feliks, Mikolaj; Field, Martin; Fieschi, Franck; Guillon, Virginia; Jakobs, Stefan; Joti, Yasumasa; Macheboeuf, Pauline; Motomura, Koji; Nass, Karol; Owada, Shigeki; Roome, Christopher M.; Ruckebusch, Cyril; Schiro, Giorgio; Shoeman, Robert L.; Thepaut, Michel; Togashi, Tadashi; Tono, Kensuke; Yabashi, Makina; Cammarata, Marco; Foucar, Lutz; Bourgeois, Dominique; Sliwa, Michel; Colletier, Jacques-Philippe; Schlichting, Ilme; Weik, MartinNature Communications (2020), 11 (1), 741CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Reversibly switchable fluorescent proteins (RSFPs) serve as markers in advanced fluorescence imaging. Photoswitching from a non-fluorescent off-state to a fluorescent on-state involves trans-to-cis chromophore isomerization and proton transfer. Whereas excited-state events on the ps timescale have been structurally characterized, conformational changes on slower timescales remain elusive. Here we describe the off-to-on photoswitching mechanism in the RSFP rsEGFP2 by using a combination of time-resolved serial crystallog. at an X-ray free-electron laser and ns-resolved pump-probe UV-visible spectroscopy. Ten ns after photoexcitation, the crystal structure features a chromophore that isomerized from trans to cis but the surrounding pocket features conformational differences compared to the final on-state. Spectroscopy identifies the chromophore in this ground-state photo-intermediate as being protonated. Deprotonation then occurs on the μs timescale and correlates with a conformational change of the conserved neighboring histidine. Together with a previous excited-state study, our data allow establishing a detailed mechanism of off-to-on photoswitching in rsEGFP2.
- 62Fu, H.; Younes, S. H. H.; Saifuddin, M.; Tepper, P. G.; Zhang, J.; Keller, E.; Heeres, A.; Szymanski, W.; Poelarends, G. J. Rapid Chemoenzymatic Route to Glutamate Transporter Inhibitor L-TFB-TBOA and Related Amino Acids. Org. Biomol. Chem. 2017, 15 (11), 2341– 2344, DOI: 10.1039/C7OB00305F62https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXjtFehu7k%253D&md5=f66db63d5a1f2e026f7b8a49b0e31c48Rapid chemoenzymatic route to glutamate transporter inhibitor L-TFB-TBOA and related amino acidsFu, Haigen; Younes, Sabry H. H.; Saifuddin, Mohammad; Tepper, Pieter G.; Zhang, Jielin; Keller, Erik; Heeres, Andre; Szymanski, Wiktor; Poelarends, Gerrit J.Organic & Biomolecular Chemistry (2017), 15 (11), 2341-2344CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)The complex amino acid (L-threo)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (L-TFB-TBOA) and its derivs. are privileged compds. for studying the roles of excitatory amino acid transporters (EAATs) in regulation of glutamatergic neurotransmission, animal behavior, and in the pathogenesis of neurol. diseases. The wide-spread use of L-TFB-TBOA stems from its high potency of EAAT inhibition and the lack of off-target binding to glutamate receptors. However, one of the main challenges in the evaluation of L-TFB-TBOA and its derivs. is the laborious synthesis of these compds. in stereoisomerically pure form. Here, we report an efficient and step-economic chemoenzymic route that gives access to enantio- and diastereopure L-TFB-TBOA and its derivs. at multigram scale.
- 63Raj, H.; Szymański, W.; De Villiers, J.; Rozeboom, H. J.; Veetil, V. P.; Reis, C. R.; De Villiers, M.; Dekker, F. J.; De Wildeman, S.; Quax, W. J.; Thunnissen, A. M. W. H.; Feringa, B. L.; Janssen, D. B.; Poelarends, G. J. Engineering Methylaspartate Ammonia Lyase for the Asymmetric Synthesis of Unnatural Amino Acids. Nat. Chem. 2012, 4 (6), 478– 484, DOI: 10.1038/nchem.133863https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xmt1Gktb8%253D&md5=e8fc35523cd38b2cb130ac8fc4813b1bEngineering methylaspartate ammonia lyase for the asymmetric synthesis of unnatural amino acidsRaj, Hans; Szymanski, Wiktor; de Villiers, Jandre; Rozeboom, Henriette J.; Veetil, Vinod Puthan; Reis, Carlos R.; de Villiers, Marianne; Dekker, Frank J.; de Wildeman, Stefaan; Quax, Wim J.; Thunnissen, Andy-Mark W. H.; Feringa, Ben L.; Janssen, Dick B.; Poelarends, Gerrit J.Nature Chemistry (2012), 4 (6), 478-484CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)The redesign of enzymes to produce catalysts for a predefined transformation remains a major challenge in protein engineering. Here, we describe the structure-based engineering of methylaspartate ammonia lyase (which in nature catalyzes the conversion of 3-methylaspartate to ammonia and 2-methylfumarate) to accept a variety of substituted amines and fumarates and catalyze the asym. synthesis of aspartic acid derivs. We obtained two single-active-site mutants, one exhibiting a wide nucleophile scope including structurally diverse linear and cyclic alkylamines and one with broad electrophile scope including fumarate derivs. with alkyl, aryl, alkoxy, aryloxy, alkylthio and arylthio substituents at the C2 position. Both mutants have an enlarged active site that accommodates the new substrates while retaining the high stereo- and regioselectivity of the wild-type enzyme. As an example, we demonstrate a highly enantio- and diastereoselective synthesis of threo-3-benzyloxyaspartate (an important inhibitor of neuronal excitatory glutamate transporters in the brain).
- 64Cheng, B.; Shchepakin, D.; Kavanaugh, M. P.; Trauner, D. Photoswitchable Inhibitor of a Glutamate Transporter. ACS Chem. Neurosci. 2017, 8 (8), 1668– 1672, DOI: 10.1021/acschemneuro.7b0007264https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmtFCmuro%253D&md5=7a430f138b3da62ba24ff4ceb2f7a681Photoswitchable Inhibitor of a Glutamate TransporterCheng, Bichu; Shchepakin, Denis; Kavanaugh, Michael P.; Trauner, DirkACS Chemical Neuroscience (2017), 8 (8), 1668-1672CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)Excitatory amino acid transporters clear glutamate from the synaptic cleft and play a crit. role in glutamatergic neurotransmission. Their differential roles in astrocytes, microglia, and neurons are poorly understood due in part to a lack of pharmacol. tools that can be targeted to specific cells and tissues. We now describe a photoswitchable inhibitor, termed ATT, that interacts with the major mammalian forebrain transporters EAAT1-3 in a manner that can be reversibly switched between trans (high-affinity) and cis (low-affinity) configurations using light of different colors. In the dark, ATT competitively inhibited the predominant glial transporter EAAT2 with ∼200-fold selectivity over the neuronal transporter EAAT3. Brief exposure to 350 nm light reduced the steady-state blocker affinity by more than an order of magnitude. Illumination of EAAT2 complexed with ATT induced a corresponding increase in the blocker off-rate monitored in the presence of glutamate. ATT can be used to reversibly manipulate glutamate transporter activity with light and may be useful to gain insights into the dynamic physiol. roles of glutamate transporters in the brain, as well as to study the mol. interactions of transporters with ligands.
- 65Morstein, J.; Awale, M.; Reymond, J. L.; Trauner, D. Mapping the Azolog Space Enables the Optical Control of New Biological Targets. ACS Cent. Sci. 2019, 5 (4), 607– 618, DOI: 10.1021/acscentsci.8b0088165https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXks1Witro%253D&md5=4a799a927450bddb1770f7801282ae94Mapping the Azolog Space Enables the Optical Control of New Biological TargetsMorstein, Johannes; Awale, Mahendra; Reymond, Jean-Louis; Trauner, DirkACS Central Science (2019), 5 (4), 607-618CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)Photopharmacol. relies on mols. that change their biol. activity upon irradn. Many of these are derived from known drugs by replacing their core with an isosteric azobenzene photoswitch (azologization). The question is how many of the known bioactive ligands could be addressed in such a way. Here, the authors systematically assess the space of mols. amenable to azologization from databases of bioactive mols. (DrugBank, PDB, CHEMBL) and the Cambridge Structural Database. Shape similarity scoring functions (3DAPfp) and analyses of dihedral angles were employed to quantify the structural homol. between a bioactive mol. and the cis or trans isomer of its corresponding azolog ("azoster") and assess which isomer is likely to be active. The authors' anal. suggests that a very large no. of bioactive ligands (>40,000) is amenable to azologization and that many new biol. targets could be addressed with photopharmacol. N-Aryl benzamides, 1,2-diarylethanes, and benzyl Ph ethers are particularly suited for this approach, while benzylanilines and sulfonamides appear to be less well-matched. On the basis of the authors' anal., the majority of azosters are expected to be active in their trans form. The broad applicability of the authors' approach is demonstrated with photoswitches that target a nuclear hormone receptor (RAR) and a lipid processing enzyme (LTA4 hydrolase).
- 66Hoorens, M. W. H.; Ourailidou, M. E.; Rodat, T.; van der Wouden, P. E.; Kobauri, P.; Kriegs, M.; Peifer, C.; Feringa, B. L.; Dekker, F. J.; Szymanski, W. Light-Controlled Inhibition of BRAFV600E Kinase. Eur. J. Med. Chem. 2019, 179, 133– 146, DOI: 10.1016/j.ejmech.2019.06.04266https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXht1Oqt7nI&md5=8fd9228db18dba5bb1a0a47f92aad8e7Light-controlled inhibition of BRAFV600E kinaseHoorens, Mark W. H.; Ourailidou, Maria E.; Rodat, Theo; van der Wouden, Petra E.; Kobauri, Piermichele; Kriegs, Malte; Peifer, Christian; Feringa, Ben L.; Dekker, Frank J.; Szymanski, WiktorEuropean Journal of Medicinal Chemistry (2019), 179 (), 133-146CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)Metastatic melanoma is amongst the most difficult types of cancer to treat, with current therapies mainly relying on the inhibition of the BRAFV600E mutant kinase. However, systemic inhibition of BRAF by small mol. drugs in cancer patients results - paradoxically - in increased wild-type BRAF activity in healthy tissue, causing side-effects and even the formation of new tumors. Here we show the development of BRAFV600E kinase inhibitors of which the activity can be switched on and off reversibly with light, offering the possibility to overcome problems of systemic drug activity by selectively activating the drug at the desired site of action. Based on a known inhibitor, eight photoswitchable effectors contg. an azobenzene photoswitch were designed, synthesized and evaluated. The most promising inhibitor showed an approx. 10-fold increase in activity upon light-activation. This research offers inspiration for the development of therapies for metastatic melanoma in which tumor tissue is treated with an active BRAFV600E inhibitor with high spatial and temporal resoln., thus limiting the damage to other tissues.
- 67Xu, Z.; Shi, L.; Jiang, D.; Cheng, J.; Shao, X.; Li, Z. Azobenzene Modified Imidacloprid Derivatives as Photoswitchable Insecticides: Steering Molecular Activity in a Controllable Manner. Sci. Rep. 2015, 5, 1– 8, DOI: 10.1038/srep13962There is no corresponding record for this reference.
- 68Kabsch, W. Integration, Scaling, Space-Group Assignment and Post-Refinement. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66 (2), 133– 144, DOI: 10.1107/S090744490904737468https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhs1Sisb4%253D&md5=eb1db34f3c959b4921771eb9f57b2159Integration, scaling, space-group assignment and post-refinementKabsch, WolfgangActa Crystallographica, Section D: Biological Crystallography (2010), 66 (2), 133-144CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)Important steps in the processing of rotation data are described that are common to most software packages. These programs differ in the details and in the methods implemented to carry out the tasks. Here, the working principles underlying the data-redn. package XDS are explained, including the new features of automatic detn. of spot size and reflecting range, recognition and assignment of crystal symmetry and a highly efficient algorithm for the detn. of correction/scaling factors.
- 69McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M. D.; Storoni, L. C.; Read, R. J. Phaser Crystallographic Software. J. Appl. Crystallogr. 2007, 40 (4), 658– 674, DOI: 10.1107/S002188980702120669https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXnslWqsLk%253D&md5=c63b722ae97e0a74e6a5a079d388f09fPhaser crystallographic softwareMcCoy, Airlie J.; Grosse-Kunstleve, Ralf W.; Adams, Paul D.; Winn, Martyn D.; Storoni, Laurent C.; Read, Randy J.Journal of Applied Crystallography (2007), 40 (4), 658-674CODEN: JACGAR; ISSN:0021-8898. (International Union of Crystallography)Phaser is a program for phasing macromol. crystal structures by both mol. replacement and exptl. phasing methods. The novel phasing algorithms implemented in Phaser have been developed using max. likelihood and multivariate statistics. For mol. replacement, the new algorithms have proved to be significantly better than traditional methods in discriminating correct solns. from noise, and for single-wavelength anomalous dispersion exptl. phasing, the new algorithms, which account for correlations between F+ and F-, give better phases (lower mean phase error with respect to the phases given by the refined structure) than those that use mean F and anomalous differences ΔF. One of the design concepts of Phaser was that it be capable of a high degree of automation. To this end, Phaser (written in C++) can be called directly from Python, although it can also be called using traditional CCP4 keyword-style input. Phaser is a platform for future development of improved phasing methods and their release, including source code, to the crystallog. community.
- 70Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Features and Development of Coot. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66 (4), 486– 501, DOI: 10.1107/S090744491000749370https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXksFKisb8%253D&md5=67262cbfc60004de5ef962d5c043c910Features and development of CootEmsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K.Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (4), 486-501CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)Coot is a mol.-graphics application for model building and validation of biol. macromols. The program displays electron-d. maps and at. models and allows model manipulations such as idealization, real-space refinement, manual rotation/translation, rigid-body fitting, ligand search, solvation, mutations, rotamers and Ramachandran idealization. Furthermore, tools are provided for model validation as well as interfaces to external programs for refinement, validation and graphics. The software is designed to be easy to learn for novice users, which is achieved by ensuring that tools for common tasks are 'discoverable' through familiar user-interface elements (menus and toolbars) or by intuitive behavior (mouse controls). Recent developments have focused on providing tools for expert users, with customisable key bindings, extensions and an extensive scripting interface. The software is under rapid development, but has already achieved very widespread use within the crystallog. community. The current state of the software is presented, with a description of the facilities available and of some of the underlying methods employed.
- 71Afonine, P. V.; Grosse-Kunstleve, R. W.; Echols, N.; Headd, J. J.; Moriarty, N. W.; Mustyakimov, M.; Terwilliger, T. C.; Urzhumtsev, A.; Zwart, P. H.; Adams, P. D. Towards Automated Crystallographic Structure Refinement with Phenix.Refine. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2012, 68 (4), 352– 367, DOI: 10.1107/S090744491200130871https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xlt1Ggu7c%253D&md5=1cc7da2cea396f1a75646ea709140cf1Towards automated crystallographic structure refinement with phenix.refineAfonine, Pavel V.; Grosse-Kunstleve, Ralf W.; Echols, Nathaniel; Headd, Jeffrey J.; Moriarty, Nigel W.; Mustyakimov, Marat; Terwilliger, Thomas C.; Urzhumtsev, Alexandre; Zwart, Peter H.; Adams, Paul D.Acta Crystallographica, Section D: Biological Crystallography (2012), 68 (4), 352-367CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)Phenix.refine is a program within the PHENIX package that supports crystallog. structure refinement against exptl. data with a wide range of upper resoln. limits using a large repertoire of model parameterizations. It has several automation features and is also highly flexible. Several hundred parameters enable extensive customizations for complex use cases. Multiple user-defined refinement strategies can be applied to specific parts of the model in a single refinement run. An intuitive graphical user interface is available to guide novice users and to assist advanced users in managing refinement projects. X-ray or neutron diffraction data can be used sep. or jointly in refinement. phenix.refine is tightly integrated into the PHENIX suite, where it serves as a crit. component in automated model building, final structure refinement, structure validation and deposition to the wwPDB. This paper presents an overview of the major phenix.refine features, with extensive literature refs. for readers interested in more detailed discussions of the methods.
Supporting Information
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Tables S1 and S2; Figures S1–S4, and information about synthesis (PDF)
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