The present study aimed at the development of fluorescent inhibitors addressing the GABA transporters mGAT1–mGAT4 as potential tool compounds in fluorescence based biological assays. The design of these fluorescent GAT inhibitors followed the structural motifs common for many GAT1–GAT4 inhibitors publicly known except that the lipophilic domain present in this compounds was replaced by a BODIPY moiety to serve as a fluorescent subunit. The fluorescent compounds obtained that way were tested for their inhibitory potencies and subtype selectivities at the four murine GABA transporter subtypes mGAT1–mGAT4 and for their binding affinity for mGAT1. All BODIPY derivatives displayed only low inhibitory potencies and subtype selectivities at the GABA transport proteins mGAT1–mGAT4, as well as low affinities for mGAT1. Still, compounds were found with reasonable binding affinities towards mGAT1 (pKi ~ 5.0) and inhibitory potencies at mGAT2 and mGAT4 (pIC50 ~ 5.0).
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Andersen KE, Begtrup M, Chorghade MS, Lee EC, Lau J, Lundt BF, Petersen H, Sørensen PO, Thøgersen H (1994) The synthesis of novel GABA uptake inhibitors. Part 2. Synthesis of 5-hydroxytiagabine, a human metabolite of the GABA reuptake inhibitor tiagabine. Tetrahedron 50:8699–8710
Andersen KE, Braestrup C, Groenwald FC, Joergensen AS, Nielsen EB, Sonnewald U, Soerensen PO, Suzdak PD, Knutsen LJS (1993) The synthesis of novel GABA uptake inhibitors. 1. Elucidation of the structure-activity studiesleading to the choice of (R)-1-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3-piperidinecarboxylic acid (Tiagabine) as an anticonvulsant drug candidate. J Med Chem 36:1716–1725
Andersen KE, Sørensen JL, Huusfeldt PO, Knutsen LJS, Lau J, Lundt BF, Petersen H, Suzdak PD, Swedberg MDB (1999) Synthesis of novel GABA uptake inhibitors. 4.1 bioisosteric transformation and successive optimization of known GABA uptake inhibitors leading to a series of potent anticonvulsant drug candidates. J Med Chem 42:4281–4291
Andersen KE, Sørensen JL, Lau J, Lundt BF, Petersen H, Huusfeldt PO, Suzdak PD, Swedberg MDB (2001) Synthesis of novel γ-aminobutyric acid (GABA) uptake inhibitors. 5.1 preparation and structure−activity studies of tricyclic analogues of known GABA uptake inhibitors. J Med Chem 44:2152–2163
Borden LA, Dhar TGM, Smith KE, Weinshank RL, Branchek TA, Gluchowski C (1994) Tiagabine, SK&F 89976-A, CI-966, and NNC-711 are selective for the cloned GABA transporter GAT-1. Eur J Pharmacol 269:219–224
Borden LA, Smith KE, Gustafson EL, Branchek TA, Weinshank RL (1995) Cloning and expression of a betaine/GABA transporter from human brain. J Neurochem 64:977–984
Briddon SJ, Hill SJ (2007) Pharmacology under the microscope: the use of fluorescence correlation spectroscopy to determine the properties of ligand-receptor complexes. Trends Pharm Sci 28:637–645
Bröer S, Gether U (2012) The solute carrier 6 family of transporters. Br J Pharmacol 167:256–278
Cai M, Varga EV, Stankova M, Mayorov A, Perry JW, Yamamura HI, Trivedi D, Hruby VJ (2006) Cell signaling and trafficking of human melanocortin receptors in real time using two-photon fluorescence and confocal laser microscopy: differentiation of agonists and antagonists. Chem Biol Drug Des 68:183–193
Chandra S, Kable EP, Morrison GH, Webb WW (1991) Calcium sequestration in the golgi apparatus of cultured mammalian cells revealed by laser scanning confocal microscopy and ion microscopy. J Cell Sci 100:747–752
Daerr M, Pabel J, Höfner G, Mayer P, Wanner KT (2019) Synthesis and biological evaluation of GAT-ligands based on meso-substituted BODIPY-dyes. Med Chem Res. https://doi.org/10.1007/s00044-019-02483-6
Elder AD, Domin A, Kaminski Schierle GS, Lindon C, Pines J, Esposito A, Kaminski CF (2009) A quantitative protocol for dynamic measurements of protein interactions by Förster resonance energy transfer-sensitized fluorescence emission. J R Soc Interface 6:S59–S81
Haradahira T, Maeda J, Okauchi T, Zhang M-R, Hojo J, Kida T, Arai T, Yamamoto F, Sasaki S, Maeda M, Suzuki K, Suhara T (2002) Synthesis, in vitro and in vivo pharmacology of a C-11 labeled analog of CP-101,606,(±)threo-1-(4-hydroxyphenyl)-2-[4-hydroxy-4-(p-[11C]methoxyphenyl)piperidino]-1-propanol, as a PET tracer for NR2B subunit-containing NMDA receptors. Nucl Med Biol 29:517–525
Jin X, Galvan A, Wichman T, Smith Y (2011) Localization and function of GABA transporters GAT-1 and GAT-3 in the basal ganglia. Front Syst Neurosci 5:63
Knutsen LJS, Andersen KE, Lau J, Lundt BF, Henry RF, Morton HE, Nærum L, Petersen H, Stephensen H, Suzdak PD, Swedberg MDB, Thomsen C, Sørensen PO (1999) Synthesis of novel GABA uptake inhibitors. 3. Diaryloxime and diarylvinyl ether derivatives of nipecotic acid and guvacine as anticonvulsant agents1. J Med Chem 42:3447–3462
Kragler A, Höfner G, Wanner KT (2008) Synthesis and biological evaluation of aminomethylphenol derivatives as inhibitors of the murine GABA transporters mGAT1–mGAT4. Eur J Med Chem 43:2404–2411
Leen V, Braeken E, Luckermans K, Jackers C, Van der Auweraer M, Boens Nl, Dehaen W (2009) A versatile, modular synthesis of monofunctionalized BODIPY dyes. Chem Commun 30:4515–4517
Leopoldo M, Lacivita E, Berardi F, Perrone R (2009) Developments in fluorescent probes for receptor research. Drug Discov Today 14:706–712
Lilley DMJ, Wilson TJ (2000) Fluorescence resonance energy transfer as a structural tool for nucleic acids. Curr Opin Chem Biol 4:507–517
Loudet A, Burgess K (2007) BODIPY dyes and their derivatives: syntheses and spectroscopic properties. Chem Rev 107:4891–4932
Lutz T, Wein T, Höfner G, Wanner KT (2017) Development of highly potent GAT1 inhibitors: synthesis of nipecotic acid derivatives with N-arylalkynyl substituents Chem. Med. Chem 12:362–371
Minelli A, DeBiasi S, Brecha NC, Vitellaro Zuccarello L, Conti F (1996) GAT-3, a high-affinity GABA plasma membrane transporter, is localized to astrocytic processes, and it is not confined to the vicinity of GABAergic synapses in the cerebral cortex. J Neurosci 16:6255
Nouel D, Gaudriault G, Houle M, Reisine T, Vincent J-P, Mazella J, Beaudet A (1997) Differential internalization of somatostatin in COS-7 cells transfected with SST1 and SST2 receptor subtypes: a confocal microscopic study using novel fluorescent somatostatin derivatives1. Endocrinology 138:296–306
Palacín M, Estévez R, Bertran J, Zorzano A (1998) Molecular biology of mammalian plasma membrane amino acid transporters. Physiol Rev 78:969–1054
Petrera M, Wein T, Allmendinger L, Sindelar M, Pabel J, Höfner G, Wanner KT (2016) Development of highly potent GAT1 inhibitors: synthesis of nipecotic acid derivatives by Suzuki–Miyaura cross-coupling reactions. Chem. Med. Chem 11:519–538
Roush WR, Kageyama M, Riva R, Brown BB, Warmus JS, Moriarty KJ (1991) Enantioselective synthesis of the bottom half of chlorothricolide. 3. Studies of the steric directing group strategy for stereocontrol in intramolecular Diels-Alder reactions. J Org Chem 56:1192–1210
Roush WR, Moriarty KJ, Brown BB (1990) Stereoselective synthesis of (Z,E)-2-bromo-1,3-dienes via the palladium (O) catalyzed cross coupling reactions of 1,1-dibromoolefins and vinylboronic acids. Tetrahedron Lett 31:6509–6512
Sculimbrene BR, Imperiali B (2006) Lanthanide-binding tags as luminescent probes for studying protein interactions. J Am Chem Soc 128:7346–7352
Serrano AL, Bilsel O, Feng G (2012) Native state conformational heterogeneity of HP35 revealed by time-resolved FRET. J Phys Chem B 116:10631–10638
Sohail A, Jayaraman K, Venkatesan S, Gotfryd K, Daerr M, Gether U, Loland CJ, Wanner KT, Freissmuth M, Sitte HH, Sandtner W, Stockner T (2016) The environment shapes the inner vestibule of LeuT. PLOS Comput Biol 12:e1005197
Still WC, Kahn M, Mitra A (1978) Rapid chromatographic technique for preparative separations with moderate resolution. J Org Chem 43:2923–2925
Tarasova NI, Stauber RH, Choi JK, Hudson EA, Czerwinski G, Miller JL, Pavlakis GN, Michejda CJ, Wank SA (1997) Visualization of G protein-coupled receptor trafficking with the aid of the green fluorescent protein: endocytosis and recycling of cholecystokinin receptor type A. J Biol Chem 272:14817–14824
Thomsen C, Sørensen PO, Egebjerg J (1997) 1-(3-(9H-Carbazol-9-yl)-1-propyl)-4-(2-methoxyphenyl)-4-piperidinol, a novel subtype selective inhibitor of the mouse type II GABA-transporter. Br J Pharmacol 120:983–985
Zepperitz C, Höfner G, Wanner KT (2006) MS-Binding Assays: kinetic, saturation, and competitive experiments based on quantitation of bound marker as exemplified by the GABA transporter mGAT1. Chem. Med. Chem 1:208–217
Zhou Y, Holmseth S, Guo C, Hassel B, Höfner G, Huitfeldt HS, Wanner KT, Danbolt NC (2012a) Deletion of the γ-aminobutyric acid transporter 2 (GAT2 and SLC6A13) gene in mice leads to changes in liver and brain taurine contents. J Biol Chem 287:35733–35746
Zhou Y, Holmseth S, Hua R, Lehre AC, Olofsson AM, Poblete-Naredo I, Kempson SA, Danbolt NC (2012b) The betaine-GABA transporter (BGT1, slc6a12) is predominantly expressed in the liver and at lower levels in the kidneys and at the brain surface. Am J Physiol Ren Physiol 302:F316–F328
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Daerr, M., Allmendinger, L., Höfner, G. et al. Synthesis and biological evaluation of fluorescent GAT-ligands based on asymmetric substituted BODIPY dyes. Med Chem Res (2020). https://doi.org/10.1007/s00044-020-02521-8
- Fluorescent ligand
- GABA transporters
- GABA uptake
- Biological activity