Photoswitchable Inhibitor of a Glutamate Transporter - ACS Chemical


Apr 17, 2017 - We now describe a photoswitchable inhibitor, termed ATT, that interacts with the major mammalian forebrain transporters EAAT1–3 in a ...
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A Photoswitchable Inhibitor of a Glutamate Transporter Bichu Cheng, Dirk Trauner, Dennis Shchepakin, and Michael P. Kavanaugh ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00072 • Publication Date (Web): 17 Apr 2017 Downloaded from http://pubs.acs.org on April 18, 2017

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A Photoswitchable Inhibitor of a Glutamate Transporter Bichu Cheng,†# Denis Shchepakin,‡# Michael P. Kavanaugh‡* and Dirk Trauner†* † ‡

LMU Munich, Department of Chemistry and Center for Integrated Protein Science (CIPSM), 81377 Munich, Germany University of Montana Center for Neuroscience, 32 Campus Drive, Missoula, MT 59803 USA

Abstract: Excitatory amino acid transporters clear glutamate from the synaptic cleft and play a critical role in glutamatergic neurotransmission. Their differential roles in astrocytes, microglia, and neurons are poorly understood due in part to a lack of pharmacological 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 physiological roles of glutamate transporters in the brain, as well as to study the molecular interactions of transporters with ligands.

Keywords: photopharmacology • neurotransmitter transporters EAAT • TBOA • glutamate

Introduction Excitatory amino acid transporters (EAATs) play an essential role in the mammalian central nervous system. They terminate the postsynaptic action of glutamate by rapidly removing released neurotransmitter from the synaptic cleft and limit its excitotoxic effect. To date, five different subtypes have been identified in mammals, namely EAAT1-5.1-3 In human forebrain, the major neuronal transporter is EAAT3 and the major glial transporter is EAAT2.4 Due to their importance in synaptic transmission and glutamate homeostasis, the EAATs have been subject to intense pharmacological studies, which has given rise to several more or less selective inhibitors.5−7 Threo-β-hydroxyaspartate (THA, 1) was discovered in the 1970s as a strong competitive inhibitor of L-glutamate uptake in rat brain slices8 and was later identified as a blocker of the rodent transporter EAAC1.9 Subsequently, Shimamoto and co-workers developed a series of L-THA derivatives that are potent inhibitors of EAAT1−3. First, they identified L-threo-β-benzyloxyaspartic acid (L-TBOA, 2) as a nontransportable blocker with IC50 values in the low micromolar range.10 L-TBOA serves as an important tool for the investigation of the physiological role of these transporters.11 It was recently cocrystallized with an aspartate transporter homologue GltPh from Pyrococcus horikoshii (pdb 2NWW), providing insight into the binding pose of these inhibitors.12 Introducing substituents at the phenyl ring of TBOA increased its activity, as exemplified by the most potent compound know to date, TFB-TBOA (3), which possesses an IC50 value in the low nanomolar range.13

Figure 1. Chemical structure of EAAT inhibitors L-THA (1), L-TBOA (2) and TFB-TBOA (3).

In recent years, our group has succeeded in developing a range of synthetic azobenzene photoswitches that can confer light sensitivity to biological systems.14, 15 They can function either as freely diffusible photochromic ligands (PCLs), or as photoswitchable tethered

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ligands (PTLs), and most recently as photoswitchable orthogonal remotely tethered ligand (PORTL). These photopharmacology techniques have placed ion channels, G-protein coupled receptors (GPCRs), enzymes, and even lipids, under the precise spatiotemporal control of light. They have found useful application in the precise control of synaptic activity and neuronal excitability. Very recently, the Wanner group has introduced an azobenzene photoswitch that targets the γ-aminobutyric acid transporter 1 (GAT1).16 We now describe the development of a photoswitchable azobenzene derivative of TFB-TBOA, termed Azo-TFB-TBOA (ATT, 4), that allows for the precise and reversible control of EAAT activity with light. As such, it provides a new way for determining the synaptic concentrations of an excitatory neurotransmitter.

Results and Discussion ATT was designed via ‘azologization’ of TFB-TBOA.14 This requires the replacement of the diaryl amide functional group by a photoisomerizable diazene (N=N) moiety. To gain rapid and reliable access to ATT, we developed a short and convergent asymmetric synthesis that could also be adapted to other TBOA analogues. Previous syntheses of TBOA (2) and TFB-TBOA (3) are either lengthy,17, 18 require an enzyme catalyst that is not readily available and works only with limited substrate scope,19 or produce the product in low enantiomeric purity.20 Our synthesis began from readily available D-diethyl tartrate (5). Treatment of 5 with HBr in acetic acid (HOAc) afforded the corresponding bromoacetate. Acetate hydrolysis in acidic ethanol and bromide displacement with sodium azide in N,Ndimethylformamide (DMF) gave the alcohol (6) in 83% yield with 5:1 dr over 3 steps.21, 22 The azobenzene fragment (9) was prepared in good yield via a Mills reaction of aniline 7 and nitrosobenzene 8 and a sequential Appel reaction. The coupling between fragments 6 and 9 was then investigated. Under basic conditions, attempted O-alkylation afforded a complicated mixture of products. However, with freshly prepared silver oxide (Ag2O), the desired product 10 was obtained in quantitative yield. The azide group was then reduced to the amine under Staudinger conditions. The two diastereomers were separable by flash chromatography (silica gel) at this stage. Finally, after hydrolysis of the ester in alkaline solution and evaporation of the organic solvent, the product ATT (4) precipitated as a hydrochloride salt from the aqueous solution upon acidification (Scheme 1).

Scheme 1. Convergent and asymmetric synthesis of ATT (4).

In its dark-adapted state, ATT (4) existed in its thermally stable trans-configuration. ATT (4) behaved as a “regular” azobenzene and could be isomerized between its trans- and cis- configurations with blue (λ = 450 nm, τ = 1.93 min) and UV-A (λ = 350 nm, τ = 2.58 min) light, respectively. Photoisomerization could be repeated over many cycles without obvious fatigue, as could be expected with an azobenzene switch. In its cis form, ATT was also semi-stable in the dark and relaxed to the trans form slowly (τ = 146 min in PBS solution) (Scheme 2).

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Scheme 2. Photoisomerization of ATT (4) in a PBS solution (5 µM). A) Light-induced isomerization monitored by UV/Vis spectroscopy. B) Repeated cycles of trans-cis isomerization of ATT with blue (λ = 450 nm) and UV-A (λ = 350 nm) light, and thermal relaxation.

With ample supplies of ATT (4) in hand, we proceeded to test its action on various excitatory amino acid transporters. Because glutamate uptake is stoichiometrically coupled to the flux of three sodium ions and one proton, with counter-flux of one potassium ion, an electrical current results during glutamate uptake that can be measured under voltage clamp conditions.23 We examined the actions of ATT on glutamate transport currents in Xenopus laevis oocytes previously injected with cRNA encoding human transporters EAAT1-3.24 Perfusion of a sub-saturating concentration L-glutamate (30 µM) induced inward currents that were reversibly blocked by co-application of ATT in the absence of light (Figure 2A). The inhibition of L-glutamate transport currents by ATT revealed subtype-selective effects of the blocker, with an order of affinity EAAT2>EAAT1>EAAT3. The ATT concentration-dependence for steady-state inhibition of 30 µM L-glutamate transport currents was determined by fitting data to inhibition isotherms as described in the Experimental Section (Figure 2B). The corresponding IC50 values of ATT (nM) by EAAT2, EAAT1, and EAAT3 were 0.9±0.1, 8.1±1.1, and 341.2±19.5nM, respectively. Using the Cheng-Prusoff equation to obtain estimated Ki values based on the IC50 values and EAAT1-3 glutamate Km values (25), we obtained Ki estimates of 0.3 nM for EAAT2, 3.2 nM for EAAT1, and 165 nM for EAAT3. ATT did not induce a current in the absence of L-glutamate in oocytes expressing any of the EAATs (see figure 4).

Figure 2. Voltage clamp recordings of currents in oocytes expressing glutamate transporters EAAT1-3 reveal subtype-selective block by ATT in the absence of light. A) Representative oocyte expressing EAAT3 showing reversible block by dark-adapted trans-ATT (1µM) of current induced by 30 µM L-glutamate. Bars above trace indicate time of perfusion of substrate and blocker. B) Concentration-dependence of steady-state inhibition of transport currents by trans-ATT yielded IC50 values (nM) for

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EAAT1 (circles), EAAT2 (triangles) and EAAT3 (squares) of 8.1±1.1, 0.9±0.1, and 341.2±19.5nM, respectively (mean ± SEM of n>4 oocytes for each subtype; holding potential = –60 mV; all recordings performed in dark conditions).

To examine the effect of photoisomerization of ATT on its glutamate transporter binding affinity, a bright monochromatic 350 nm light source was introduced by fiber optic into the perfusion channel of the recording chamber just upstream of the cell, and the steady-state ATT inhibition of currents induced by 30 µM L-glutamate was recorded in the presence or absence of light. In the presence of 350 nm light, the concentration-dependence of inhibition of transport currents mediated by EAAT1-3 were right-shifted between 3 and 14-fold, to 66.4±4.9nM, 12.7±1.4 nM, and 1132 ± 51 nM respectively (n=5; Figure 3).

Figure 3. Steady-state ATT concentration-dependence of block of transport currents induced by 30 µM L-glutamate in the absence (open symbol) or presence (filled symbol) of 350 nm light. The fitted IC50 values were shifted approximately 8,14, and 3-fold for EAAT1, EAAT2, and EAAT3, respectively. Holding potential = -60 mV.

In order to test whether the photoisomerization of ATT could also occur while the molecule was docked in the transporter binding site, we pre-incubated oocytes expressing EAAT2 with a near-saturating concentration of ATT (10 nM), and then tested the recovery of the transport current following washout of the drug in the continuous presence of 30 µM L-glutamate. In the absence of light, the transport current recovered slowly, reflecting the slow unbinding kinetics of nanomolar affinity EAAT blockers.13, 25 When pulses of 350 nm light were directed onto the surface of the oocyte during this washout phase, the current recovery kinetics switched to a fast mode (Figure 4). When the light was switched off, the remaining blocked fraction returned to a slow recovery mode, suggesting that the remaining fraction of transporters remained blocked by the high-affinity isomer (Figure 4).

Figure 4. Effects of 350 nm light on kinetics of recovery from ATT block indicate that the blocker is capable of undergoing photoisomerization while in complex with the transporter. In the dark, EAAT2-mediated currents induced by perfusion of L-glutamate (30µM) are blocked by pre-incubation with ATT (10 nM). Washout of blocker in the presence of L-glutamate (30 µM) reveals a slow recovery from block that is accelerated during 350 nm light pulse intervals indicated by blue bars. The timing of ligand perfusion is indicated by bars above the trace. In the absence of ATT, light pulses had no effect on holding currents nor glutamate-induced currents in oocytes expressing EAAT1-3 (not shown). Holding potential = –20 mV.

Conclusions In conclusion, we have developed ATT as the first photochromic inhibitor of the EAAT family of glutamate transporters. A short and asymmetric synthesis of ATT was developed, which could potentially be used to procure TBOA itself and many of new derivatives thereof. ATT can be reversibly switched between trans (high-affinity) and cis (low-affinity) configurations using light of different colors. In its darkadapted form, ATT displayed subtype-selectivity with a subnanomolar affinity for the glial EAAT2 subtype, the most abundant glutamate transporter in brain. Photoisomerization of the high-affinity trans-configuration to the cis-configuration by 350 nm light reduced ATT affinity for EAAT2 by approximately 14-fold. Photoisomerization could also be induced in docked transporter complexes within a discrete illuminated spatial domain, resulting in a corresponding increase in the blocker off-rate monitored in the presence of glutamate. The results demonstrate that ATT can be used to reversibly manipulate glutamate transporter activity with light. As such, it may be a useful tool to

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gain insights into the physiological roles of glutamate transporters in the brain, as well as to study the dynamic molecular interactions of transporters with ligands. Studies in this direction are currently underway in our laboratory.

Experimental Section Oocyte expression and recording: Xenopus laevis oocytes (Ecocyte BioScience) were injected with approximately 50 ng of human EAAT1, EAAT2, or EAAT3 cRNA (Arriza et al. 1994) and two-microelectrode voltage clamp recordings were performed 3–5 days later. Recording electrodes (0.2–1.0 MΩ) were filled with 3 M KCl. Recording solution (frog Ringer) contained (mM): 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, 5 HEPES pH 7.5. Recordings were made with a GeneClamp 500 amplifier (Molecular Devices) and an analog-digital converter (ADInstruments) interfaced to a Macintosh computer. Light was introduced into the recording chamber perfusion channel or directly onto the oocyte with a fiber-optic light guide coupled to a mononchrometer controlled by computer (Till). Data were analyzed with LabChart and Kaleidagraph software. Responses to 30 µM L-glutamate were recorded and steady-state inhibition curves to determine IC50 values of ATT were constructed by fitting current amplitude data to the expression: %inhibition = [ATT]/([ATT]+IC50). IC50 values are expressed as the mean +/- SEM in at least 4 oocytes expressing each transporter. Supporting Information Experimental procedure for the chemical synthesis of ATT AUTHOR INFORMATION Corresponding Author(s)

*E-mail: [email protected]; [email protected] Author contributions # These authors contributed equally to the work Notes The authors declare no competing financial interest.

Acknowledgements We gratefully acknowledge the European Research Council (ERC Advanced Grant 268795 to D.T.) and NIH (R15GM088789 and P20GM103546 to M.K.) for financial support. [1] [2] [3] [4] [5]

[6]

[7]

[8] [9] [10] [11]

Watkins, J. C., Jane, D. E. (2006) The glutamate story. Br. J. Pharmacol. 147, S100–S108. Beart, P. M., O’Shea, R. D. (2007) Transporters for L-glutamate: an update on their molecular pharmacology and pathological involvement. Br. J. Pharmacol. 150, 5–17. Divito, C. B., Underhill, S. M. (2014) Excitatory amino acid transporters: roles in glutamatergic neurotransmission. Neurochem. Int. 73, 172–180. Tzingounis, A. V., Wadiche, J. I. (2007) Glutamate transporters: confining runaway excitation by shaping synaptic transmission. Nat. Rev. Neurosci. 935−947. a) Jensen, A. A., Erichsen, M. N., Nielsen, C. W., Stensbøl, T. B., Kehler, J., Bunch, L. (2009) Discovery of the first selective inhibitor of excitatory amino acid transporter subtype 1. J. Med. Chem. 52, 912–915; b) Hansen, S. W.; Erichsen, M. N.; Fu, B.; Bjørn-Yoshimoto, W. E.; Abrahamsen, B.; Hansen, J. C.; Jensen, A. A.; Bunch, L. (2016) 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. 59, 8757-8770. c) Abrahamsen, B., Schneider, N., Erichsen, M. N., Huynh, T. H., Fahlke, C., Bunch, L., Jensen, A. A. (2013) 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. 33, 1068−1087. Dunlop, J.; McIlvain, H. B.; Carrick, T. A.; Jow, B.; Lu, Q.; Kowal, D.; Lin, S.; Greenfield, A.; Grosanu, C.; Fan, K.; Petroski, R.; Williams, J.; Foster, A.; Butera, J. (2005) Characterization of novel aryl-ether, biaryl, and fluorene aspartic acid and diaminopropionic acid analogs as potent inhibitors of the high-affinity glutamate transporter EAAT2. Mol. Pharmacol. 68, 974−982. Esslinger, 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. (2005) The substituted aspartate analogue L-β-threo-benzyl-aspartate preferentially inhibits the neuronal excitatory amino acid transporter EAAT3. Neuropharmacology 49, 850−861. Balcar, V. J., Johnston, G. A. (1972) The structural specificity of the high affinity uptake of L-glutamate and L-aspartate by rat brain slices. J. Neurochem. 19, 2657–2666. Kanai, Y., Hediger, M. A. (1992) Primary structure and functional characterization of a high-affinity glutamate transporter. Nature 360, 467−471. Shimamoto, K., Lebrun, B., Yasuda-Kamatani, Y., Sakaitani, M., Shigeri, Y., Yumoto, N., Nakajima, T. (1998) DL-threo-β-benzyloxyaspartate, a potent blocker of excitatory amino acid transporters. Mol. Pharmacol. 53, 195−201. Jabaudon, D., Shimamoto, K., Yasuda-Kamatani, Y., Scanziani, M., Gähwiler, B. H., Gerber, U. (1999) Inhibition of uptake unmasks rapid extracellular turnover of glutamate of nonvesicular origin. Proc. Natl. Acad. Sci. USA 96, 8733-8738.

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[12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25]

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Boudker, O., Ryan, R. M., Yernool, D., Shimamoto, K., Gouaux, E. (2007) Coupling substrate and ion binding to extracellular gate of a sodium-dependent aspartate transporter. Nature 445, 387−393. K. Shimamoto, R. Sakai, K. Takaoka, N. Yumoto, T. Nakajima, S. G. Amara, Y. Shigeri, Mol. Pharmacol. 2004, 65, 1008−1015. Broichhagen, J., Frank, J. A., Trauner, D. (2015) A roadmap to success in photopharmacology. Acc. Chem. Res. 48, 1947−1960. Fehrentz, T., Schönberger, M., Trauner, D. (2011) Optochemical genetics. Angew. Chem. Int. Ed. 50, 12156–12182. Quandt, G., Höfner, G., Pabel, J., Dine, J., Eder, M., Wanner, K. T. (2014) First photoswitchable neurotransmitter transporter inhibitor: light-induced control of γaminobutyric acid transporter 1 (GAT1) activity in mouse brain. J. Med. Chem. 57, 6809–6821. Shimamoto, K., Shigeri, Y., Yasuda-Kamatani, Y., Lebrun, B., Yumoto, N., Nakajima, T. (2000) Syntheses of optically pure β-hydroxyaspartate derivatives as glutamate transporter blockers. Bioorg. Med. Chem. Lett. 10, 2407−2410. Shimamoto, K. (2003) β-Benzyloxyaspartate derivatives with amino group on benzene ring. Patent WO2003/000698 A1. de Villiers, J., de Villiers, M., Geertsema, E. M., Raj, H., Poelarends, G. J. (2015) Chemoenzymatic synthesis of ortho-, meta-, and para-substituted derivatives of L-threo-3-benzyloxyaspartate, an important glutamate transporter blocker. Chemcatchem 7, 1931–1934. Leuenberger, M., Ritler, A., Simonin, A., Hediger, M. A., Lochner, M., (2016) Concise asymmetric synthesis and pharmacological characterization of all stereoisomers of glutamate transporter inhibitor TFB-TBOA and synthesis of EAAT photoaffinity probes. ACS Chem. Neurosci. 7, 534–539. Mori, K., Iwasawa, H. (1980) Stereoselective synthesis of optically active forms of δ-multistriatin, the attractant for European populations of the smaller European elm bark beetle. Tetrahedron 36, 87–90. France, B., Bruno, V., Nicolas, I. (2013) Synthesis of a protected derivative of (2R,3R)-β-hydroxyaspartic acid suitable for Fmoc-based solid phase synthesis. Tetrahedron Lett. 54, 158–161. Zerangue, N., Kavanaugh, M. P. (1996) Flux coupling in a neuronal glutamate transporter. Nature 383, 634–637. Arriza, J. L., Fairman, W. A., Wadiche, J. I., Murdoch, G. H., Kavanaugh, M. P., Amara, S. G. (1994) Functional comparisons of three glutamate transporter subtypes cloned from human motor cortex. J. Neurosci. 14, 5559–5569. Leary, G. P., Holley, D. C., Stone, E. F., Lyda, B. R., Kalachev, L. V., Kavanaugh, M. P. (2011) The central cavity in trimeric glutamate transporters restricts ligand diffusion. Proc. Natl. Acad. Sci. U.S.A. 108, 14980–14985.

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