Article | Published:

Discovery of allosteric modulators for GABAA receptors by ligand-directed chemistry

Nature Chemical Biology volume 12, pages 822830 (2016) | Download Citation

Abstract

The fast inhibitory actions of γ-aminobutyric acid (GABA) are mainly mediated by GABAA receptors (GABAARs) in the brain. The existence of multiple ligand-binding sites and a lack of structural information have hampered the efficient screening of drugs capable of acting on GABAARs. We have developed semisynthetic fluorescent biosensors for orthosteric and allosteric GABAAR ligands on live cells via coupling of affinity-based chemical labeling reagents to a bimolecular fluorescence quenching and recovery system. These biosensors were amenable to the high-throughput screening of a chemical library, leading to the discovery of new small molecules capable of interacting with GABAARs. Electrophysiological measurements revealed that one hit, 4,4′,4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), was a novel negative allosteric modulator capable of strongly suppressing GABA-induced chloride currents. Thus, these semisynthetic biosensors represent versatile platforms for screening drugs to treat GABAAR-related neurological disorders, and this strategy can be extended to structurally complicated membrane proteins.

  • Compound

    4-(3-(4-((4-(3-(2-(1-((2-(2-(2',7'-difluoro-3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-carboxamido)ethoxy)ethoxy)carbonyl)-1H-imidazol-4-yl)acetamido)propyl)benzyl)oxy)phenyl)-6-iminopyridazin-1(6H)-yl)butanoic acid

  • Compound

    2-(2-(2',7'-difluoro-3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-carboxamido)ethoxy)ethyl 4-(19-(7-chloro-5-(2-fluorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-1-yl)-2,16-dioxo-6,9,12-trioxa-3,15-diazanonadecyl)-1H-imidazole-1-carboxylate

  • Compound

    2-(2-(2',7'-difluoro-3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-carboxamido)ethoxy)ethyl 4-(2-((3-(4-(3-(5-ethyl-4,6-dioxo-2-thioxohexahydropyrimidin-5-yl)propyl)-1H-1,2,3-triazol-1-yl)propyl)amino)-2-oxoethyl)-1H-imidazole-1-carboxylate

  • Compound

    (Z)-N-(9-(2-((4-((3-(4-((4-(1-(3-carboxypropyl)-6-imino-1,6-dihydropyridazin-3-yl)phenoxy)methyl)phenyl)propyl)carbamoyl)piperidin-1-yl)sulfonyl)phenyl)-6-(methyl(phenyl)amino)-3H-xanthen-3-ylidene)-N-methylbenzenaminium

  • Compound

    (Z)-N-(9-(2-((4-((2-(2-(2-((3-(7-chloro-5-(2-fluorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-1-yl)propyl)amino)-2-oxoethoxy)ethoxy)ethyl)carbamoyl)piperidin-1-yl)sulfonyl)phenyl)-6-(methyl(phenyl)amino)-3H-xanthen-3-ylidene)-N-methylbenzenaminium

  • Compound

    (E)-1-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one

  • Compound

    4-(1,5-bis(4-hydroxyphenyl)-4-propyl-1,2-dihydro-3H-pyrazol-3-ylidene)cyclohexa-2,5-dien-1-one

  • Compound

    ethyl 8-fluoro-5-methyl-6-oxo-5,6-dihydro-4H-benzo[f]imidazo[1,5-α][1,4]diazepine-3-carboxylate

  • Compound

    4,5,6,7-tetrabromo-1H-benzo[d][1,2,3]triazole

  • Compound

    N-(2-(2-(2-((3-(7-chloro-5-(2-fluorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-1-yl)propyl)amino)-2-oxoethoxy)ethoxy)ethyl)-2',7'-difluoro-3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-carboxamide

  • Compound

    5,6-dibromo-1H-benzo[d][1,2,3]triazole

  • Compound

    5-bromo-1H-benzo[d][1,2,3]triazole

  • Compound

    3-(perbromo-1H-benzo[d][1,2,3]triazol-1-yl)propan-1-ol

  • Compound

    3-(perbromo-2H-benzo[d][1,2,3]triazol-2-yl)propan-1-ol

  • Compound

    tert-butyl (3-(4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)prop-2-yn-1-yl)carbamate

  • Compound

    tert-butyl (3-(4-(hydroxymethyl)phenyl)prop-2-yn-1-yl)carbamate

  • Compound

    tert-butyl (3-(4-(hydroxymethyl)phenyl)propyl)carbamate

  • Compound

    tert-butyl (3-(4-(bromomethyl)phenyl)propyl)carbamate

  • Compound

    tert-butyl (3-(4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)methyl)phenyl)propyl)carbamate

  • Compound

    tert-butyl (3-(4-((4-(6-aminopyridazin-3-yl)phenoxy)methyl)phenyl)propyl)carbamate

  • Compound

    methyl 4-(3-(4-((4-(3-((tert-butoxycarbonyl)amino)propyl)benzyl)oxy)phenyl)-6-iminopyridazin-1(6H)-yl)butanoate

  • Compound

    methyl 4-(3-(4-((4-(3-(2-(1H-imidazol-4-yl)acetamido)propyl)benzyl)oxy)phenyl)-6-iminopyridazin-1(6H)-yl)butanoate

  • Compound

    4-(3-(4-((4-(3-(2-(1H-imidazol-4-yl)acetamido)propyl)benzyl)oxy)phenyl)-6-iminopyridazin-1(6H)-yl)butanoic acid

  • Compound

    2',7'-difluoro-3',6'-dihydroxy-N-(2-(2-hydroxyethoxy)ethyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-carboxamide

  • Compound

    4-(3-(4-((4-(3-((tert-butoxycarbonyl)amino)propyl)benzyl)oxy)phenyl)-6-iminopyridazin-1(6H)-yl)butanoic acid

  • Compound

    tert-butyl (2-(2-(((perfluorophenoxy)carbonyl)oxy)ethoxy)ethyl)carbamate

  • Compound

    tert-butyl 4-(7-chloro-5-(2-fluorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-1-yl)butanoate

  • Compound

    tert-butyl (16-(7-chloro-5-(2-fluorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-1-yl)-13-oxo-3,6,9-trioxa-12-azahexadecyl)carbamate

  • Compound

    N-(1-(1H-imidazol-4-yl)-2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-4-(7-chloro-5-(2-fluorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-1-yl)butanamide

  • Compound

    2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl 4-(19-(7-chloro-5-(2-fluorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-1-yl)-2,16-dioxo-6,9,12-trioxa-3,15-diazanonadecyl)-1H-imidazole-1-carboxylate

  • Compound

    tert-butyl (3-(7-chloro-5-(2-fluorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-1-yl)propyl)carbamate

  • Compound

    (9H-fluoren-9-yl)methyl (2-(2-(2-((3-(7-chloro-5-(2-fluorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-1-yl)propyl)amino)-2-oxoethoxy)ethoxy)ethyl)carbamate

  • Compound

    diethyl 2-ethyl-2-(pent-4-yn-1-yl)malonate

  • Compound

    5-ethyl-5-(pent-4-yn-1-yl)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione

  • Compound

    tert-butyl (3-(4-(3-(5-ethyl-4,6-dioxo-2-thioxohexahydropyrimidin-5-yl)propyl)-1H-1,2,3-triazol-1-yl)propyl)carbamate

  • Compound

    N-(3-(4-(3-(5-ethyl-4,6-dioxo-2-thioxohexahydropyrimidin-5-yl)propyl)-1H-1,2,3-triazol-1-yl)propyl)-2-(1H-imidazol-4-yl)acetamide

  • Compound

    2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl 4-(2-((3-(4-(3-(5-ethyl-4,6-dioxo-2-thioxohexahydropyrimidin-5-yl)propyl)-1H-1,2,3-triazol-1-yl)propyl)amino)-2-oxoethyl)-1H-imidazole-1-carboxylate

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    et al. International Union of Pharmacology. XV. Subtypes of γ-aminobutyric acidA receptors: classification on the basis of subunit structure and receptor function. Pharmacol. Rev. 50, 291–313 (1998).

  2. 2.

    & GABA-activated ligand gated ion channels: medicinal chemistry and molecular biology. J. Med. Chem. 43, 1427–1447 (2000).

  3. 3.

    & GABAA receptors: subtypes provide diversity of function and pharmacology. Neuropharmacology. 56, 141–148 (2009).

  4. 4.

    & Beyond classical benzodiazepines: novel therapeutic potential of GABAA receptor subtypes. Nat. Rev. Drug Discov. 10, 685–697 (2011).

  5. 5.

    Allosteric modulation of GABAA receptors via multiple drug-binding sites. Adv. Pharmacol. 72, 53–96 (2015).

  6. 6.

    & Engineering biosensors by introducing fluorescent allosteric signal transducers: construction of a novel glucose sensor. J. Am. Chem. Soc. 120, 7–11 (1998).

  7. 7.

    , & Recognition-domain focused chemosensors: versatile and efficient reporters of protein kinase activity. J. Am. Chem. Soc. 130, 12821–12827 (2008).

  8. 8.

    et al. An in vivo fluorescent sensor reveals intracellular ins(1,3,4,5)P4 dynamics in single cells. Angew. Chem. Int. Edn Engl. 49, 2150–2153 (2010).

  9. 9.

    et al. Converting a solvatochromic fluorophore into a protein-based pH indicator for extreme acidity. Angew. Chem. Int. Edn Engl. 51, 7674–7679 (2012).

  10. 10.

    et al. High-throughput development of a hybrid-type fluorescent glutamate sensor for analysis of synaptic transmission. Angew. Chem. Int. Edn Engl. 53, 13439–13443 (2014).

  11. 11.

    & Recent progress in design of protein-based fluorescent biosensors and their cellular applications. ACS Chem. Biol. 9, 2708–2717 (2014).

  12. 12.

    et al. Development of a fluorescent-tagged kinase assay system for the detection and characterization of allosteric kinase inhibitors. J. Am. Chem. Soc. 131, 13286–13296 (2009).

  13. 13.

    et al. High-throughput screening to identify inhibitors which stabilize inactive kinase conformations in p38α. J. Am. Chem. Soc. 131, 18478–18488 (2009).

  14. 14.

    , , , & Selective detection of allosteric phosphatase inhibitors. J. Am. Chem. Soc. 135, 6838–6841 (2013).

  15. 15.

    & Monitoring ligand-induced conformational changes for the identification of estrogen receptor agonists and antagonists. Angew. Chem. Int. Edn Engl. 54, 4379–4382 (2015).

  16. 16.

    , , , & Semisynthetic fluorescent sensor proteins based on self-labeling protein tags. J. Am. Chem. Soc. 131, 5873–5884 (2009).

  17. 17.

    , , , & A fluorescent sensor for GABA and synthetic GABA(B) receptor ligands. J. Am. Chem. Soc. 134, 19026–19034 (2012).

  18. 18.

    et al. A semisynthetic fluorescent sensor protein for glutamate. J. Am. Chem. Soc. 134, 7676–7678 (2012).

  19. 19.

    , & Benzodiazepine receptor protein identified and visualized in brain tissue by a photoaffinity label. Proc. Natl. Acad. Sci. USA 77, 1666–1670 (1980).

  20. 20.

    et al. Identification of a GABAA receptor anesthetic binding site at subunit interfaces by photolabeling with an etomidate analog. J. Neurosci. 26, 11599–11605 (2006).

  21. 21.

    et al. Allyl m-trifluoromethyldiazirine mephobarbital: an unusually potent enantioselective and photoreactive barbiturate general anesthetic. J. Med. Chem. 55, 6554–6565 (2012).

  22. 22.

    et al. A propofol binding site on mammalian GABAA receptors identified by photolabeling. Nat. Chem. Biol. 9, 715–720 (2013).

  23. 23.

    et al. Relative positioning of classical benzodiazepines to the γ2-subunit of GABAA receptors. ACS Chem. Biol. 9, 1846–1853 (2014).

  24. 24.

    et al. Photo-antagonism of the GABAA receptor. Nat. Commun. 5, 4454 (2014).

  25. 25.

    , , , & Ligand-directed tosyl chemistry for protein labeling in vivo. Nat. Chem. Biol. 5, 341–343 (2009).

  26. 26.

    et al. Chemical cell-surface receptor engineering using affinity-guided, multivalent organocatalysts. J. Am. Chem. Soc. 133, 12220–12228 (2011).

  27. 27.

    , , , & Ligand-directed acyl imidazole chemistry for labeling of membrane-bound proteins on live cells. J. Am. Chem. Soc. 134, 3961–3964 (2012).

  28. 28.

    et al. LDAI-based chemical labeling of intact membrane proteins and its pulse-chase analysis under live cell conditions. Chem. Biol. 21, 1013–1022 (2014).

  29. 29.

    , , , & Synthesis and evaluation of highly potent GABA(A) receptor antagonists based on gabazine (SR-95531). Bioorg. Med. Chem. Lett. 21, 4252–4254 (2011).

  30. 30.

    & Structural mechanisms underlying benzodiazepine modulation of the GABA(A) receptor. J. Neurosci. 28, 3490–3499 (2008).

  31. 31.

    et al. Recognition of anesthetic barbiturates by a protein binding site: a high resolution structural analysis. PLoS One 7, e32070 (2012).

  32. 32.

    et al. Conformation of receptor adopted upon interaction with virus revealed by site-specific fluorescence quenchers and FRET analysis. J. Am. Chem. Soc. 131, 5478–5482 (2009).

  33. 33.

    , , , & Functional and molecular distinction between recombinant rat GABAA receptor subtypes by Zn2+. Neuron 5, 781–788 (1990).

  34. 34.

    , , & Zinc-mediated inhibition of GABA(A) receptors: discrete binding sites underlie subtype specificity. Nat. Neurosci. 6, 362–369 (2003).

  35. 35.

    , , & Rat β 3 subunits expressed in human embryonic kidney 293 cells form high affinity [35S]t-butylbicyclophosphorothionate binding sites modulated by several allosteric ligands of gamma-aminobutyric acid type A receptors. Mol. Pharmacol. 48, 385–391 (1995).

  36. 36.

    , & Modulation by general anaesthetics of rat GABAA receptors comprised of α 1 β 3 and β 3 subunits expressed in human embryonic kidney 293 cells. Br. J. Pharmacol. 120, 899–909 (1997).

  37. 37.

    et al. Diazepam-bound GABAA receptor models identify new benzodiazepine binding-site ligands. Nat. Chem. Biol. 8, 455–464 (2012).

  38. 38.

    et al. Specificity of intersubunit general anesthetic-binding sites in the transmembrane domain of the human α1β3γ2 γ-aminobutyric acid type A (GABAA) receptor. J. Biol. Chem. 288, 19343–19357 (2013).

  39. 39.

    , & Differences in affinity and efficacy of benzodiazepine receptor ligands at recombinant gamma-aminobutyric acidA receptor subtypes. Mol. Pharmacol. 43, 240–244 (1993).

  40. 40.

    , , & The benzodiazepine binding pocket of recombinant α1β2γ2 γ-aminobutyric acidA receptors: relative orientation of ligands and amino acid side chains. Mol. Pharmacol. 54, 1097–1105 (1998).

  41. 41.

    , & Type I and type II GABAA-benzodiazepine receptors produced in transfected cells. Science 245, 1389–1392 (1989).

  42. 42.

    Structure and pharmacology of gamma-aminobutyric acidA receptor subtypes. Pharmacol. Rev. 47, 181–234 (1995).

  43. 43.

    et al. [3H]L-655,708, a novel ligand selective for the benzodiazepine site of GABAA receptors which contain the α 5 subunit. Neuropharmacology. 35, 1331–1335 (1996).

  44. 44.

    et al. Isoliquiritigenin, a chalcone compound, is a positive allosteric modulator of GABAA receptors and shows hypnotic effects. Biochem. Biophys. Res. Commun. 413, 637–642 (2011).

  45. 45.

    et al. Pyrazole ligands: structure-affinity/activity relationships and estrogen receptor-α-selective agonists. J. Med. Chem. 43, 4934–4947 (2000).

  46. 46.

    et al. Selectivity of 4,5,6,7-tetrabromobenzotriazole, an ATP site-directed inhibitor of protein kinase CK2 ('casein kinase-2'). FEBS Lett. 496, 44–48 (2001).

  47. 47.

    , , , & GABA A/Bz receptor subtypes as targets for selective drugs. Curr. Med. Chem. 14, 2680–2701 (2007).

  48. 48.

    et al. Benzodiazepine binding site occupancy by the novel GABAA receptor subtype-selective drug 7-(1,1-dimethylethyl)-6-(2-ethyl-2H–1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine (TPA023) in rats, primates, and humans. J. Pharmacol. Exp. Ther. 332, 17–25 (2010).

  49. 49.

    et al. L-655,708 enhances cognition in rats but is not proconvulsant at a dose selective for α5-containing GABAA receptors. Neuropharmacology 51, 1023–1029 (2006).

  50. 50.

    , , & Influence of recombinant gamma-aminobutyric acid-A receptor subunit composition on the action of allosteric modulators of gamma-aminobutyric acid-gated Cl currents. Mol. Pharmacol. 39, 691–696 (1991).

  51. 51.

    et al. The clustering of GABA(A) receptor subtypes at inhibitory synapses is facilitated via the direct binding of receptor α 2 subunits to gephyrin. J. Neurosci. 28, 1356–1365 (2008).

  52. 52.

    et al. Gephyrin regulates the cell surface dynamics of synaptic GABAA receptors. J. Neurosci. 25, 10469–10478 (2005).

  53. 53.

    , & Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193–199 (1991).

  54. 54.

    , , & Molecular dissection of benzodiazepine binding and allosteric coupling using chimeric γ-aminobutyric acidA receptor subunits. Mol. Pharmacol. 53, 295–303 (1998).

  55. 55.

    & Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 22, 3099–3108 (1973).

Download references

Acknowledgements

We thank S. Moss (Tufts University) for GABAAR constructs. The pCAGGS vector was provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan. We thank Y. Yasueda (Kyoto University) and E. Kusaka (Kyoto University) for excellent technical assistance. This work was funded by the Japan Science and Technology Agency (JST) Core Research for Evolutional Science and Technology (CREST) of Molecular Technologies and JSPS KAKENHI (JP15H01637) to I.H. and by a SUNBOR Grant from Suntory Foundation for Life Sciences to S.K.

Author information

Affiliations

  1. Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto, Japan.

    • Kei Yamaura
    • , Shigeki Kiyonaka
    •  & Itaru Hamachi
  2. Department of Technology and Ecology, Hall of Global Environmental Studies, Kyoto University, Kyoto, Japan.

    • Shigeki Kiyonaka
  3. Department of Physiology, School of Medicine, Fukuoka University, Jonan-ku, Fukuoka, Japan.

    • Tomohiro Numata
    •  & Ryuji Inoue
  4. Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Kawaguchi, Saitama, Japan.

    • Itaru Hamachi

Authors

  1. Search for Kei Yamaura in:

  2. Search for Shigeki Kiyonaka in:

  3. Search for Tomohiro Numata in:

  4. Search for Ryuji Inoue in:

  5. Search for Itaru Hamachi in:

Contributions

S.K. and I.H. initiated and designed the project. K.Y. performed synthesis and chemical labeling in cultured cells. K.Y. and S.K. performed molecular biology experiments. S.K. performed radioisotope experiments. T.N. and R.I. performed electrophysiological experiments. K.Y., S.K., and I.H. wrote the manuscript. All authors discussed and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Shigeki Kiyonaka or Itaru Hamachi.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Results, Supplementary Tables 1 and 2 and Supplementary Figures 1–21.

  2. 2.

    Supplementary Note

    Synthetic Procedures

Excel files

  1. 1.

    Supplementary Data Set 1

    The entire list of pharmacologically active compounds (LOPAC1280).

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nchembio.2150

Further reading