Login Register Cart 0
Generic placeholder image

Combinatorial Chemistry & High Throughput Screening

Editor-in-Chief

ISSN (Print): 1386-2073
ISSN (Online): 1875-5402

Back
Journal Home About Journal Editorial Board Journal Insight Current Issue Volumes/Issues
Subscribe
Research Article

Establishment of a High Throughput Screening System for GABAA1 Modulators in Living Cells

Author(s): Yi Zhang, Tong Shi, Xuejun Chen, Ruihua Zhang, Jingjing Shi, Qian Jin, Jianfu Xu, Chen Wang* and Liqin Li*

Volume 26, Issue 4, 2023

Published on: 29 August, 2022

Page: [801 - 814] Pages: 14

DOI: 10.2174/1386207325666220627163438

Price: $65

Abstract

Background: The incidence of sleep disorders is more than 27% in the worldwide, and the development of novel sleep drugs that target GABAA receptors is of great interest. Traditional drug screening methods restrict the discovery of lead compounds, the high-throughput screening system is a powerful means for the lead compounds discovery of sleep drug.

Methods: The GABAA1-CHO cell line stably expressing α1β2γ2L was constituted by cotransfection of α1, β2 and γ2L subunits into CHO-T-Rex cells. The high-throughput screening method of membrane potential targeting GABAAR was established and optimized. The optimized method was used to screen the compound library, and the compounds with high activity were obtained. The active compounds were confirmed in vitro by electrophysiological detection technique, and the sleep effects of compounds in vivo were detected by pentobarbital sodium sleep model in mice.

Results: A stable cell line expressing human GABAA1 receptor in CHO-T-Rex cells was generated and used to establish a functional high-throughput screening assay based on the measurement of membrane potential changes in living cells by fluorometric imaging plate reader (FLIPR). The assay was further used to detect the dose-effect relationships of tool compounds, the EC50 values of agonist GABA (137.42 ± 26.31 nM), positive allosteric modulator diazepam (3.22 ± 0.73 μM), and antagonist gabazine (0.16 ± 0.04 μM), blocking agents bicuculine (0.47 ± 0.06 μM) and PTX (6.39 ± 1.17 μM). In the meanwhile, the compounds were screened from a compound library (10000) by the membrane potential dye assay. Selected 4 active compounds were further identified for their EC50 values in vitro by electrophysiological method, the EC50 values of 4 compounds were further determined as 1.37 ± 0.43 μM, 0.69 ± 0.17 μM, 0.77 ± 0.16 μM, and 1.62 ± 0.29 μM. Furthermore, the pentobarbital sleep rate and the sleep time of mice pretreated with 4 active compounds by oral administration were significantly increased compared with mice pretreated with a negative control in vivo experiment.

Conclusion: We successfully generated a stable CHO cell line expressing human GABAA1 by induced expression strategy which decreased cytotoxicity. Then, developed an efficient membrane potential detection method for high-throughput screening, the assay based on the stable cell line could distinguish different types of GABAA1 modulators, which would be an effective in vitro system to screen the GABAAR-targeted compounds. Compared with the patch clamp electrophysiological detection method, the membrane potential detection method has higher detection flux for compounds and higher detection sensitivity for active compounds.

Keywords: GABAA1, stable cell line, membrane potential, FLIPR, screen, high-throughput.

« Previous Next »
[1]
Brickley, S.G.; Mody, I. Extrasynaptic GABA(A) receptors: Their function in the CNS and implications for disease. Neuron, 2012, 73(1), 23-34.
[http://dx.doi.org/10.1016/j.neuron.2011.12.012] [PMID: 22243744]
[2]
Nickolls, S.A.; Gurrell, R.; van Amerongen, G.; Kammonen, J.; Cao, L.; Brown, A.R.; Stead, C.; Mead, A.; Watson, C.; Hsu, C.; Owen, R.M.; Pike, A.; Fish, R.L.; Chen, L.; Qiu, R.; Morris, E.D.; Feng, G.; Whitlock, M.; Gorman, D.; van Gerven, J.; Reynolds, D.S.; Dua, P.; Butt, R.P. Pharmacology in translation: The preclinical and early clinical profile of the novel α2/3 functionally selective GABAA receptor positive allosteric modulator PF-06372865. Br. J. Pharmacol., 2018, 175(4), 708-725.
[http://dx.doi.org/10.1111/bph.14119] [PMID: 29214652]
[3]
Seljeset, S.; Laverty, D.; Smart, T.G. Inhibitory neurosteroids and the GABAA receptor. Adv. Pharmacol., 2015, 72, 165-187.
[http://dx.doi.org/10.1016/bs.apha.2014.10.006] [PMID: 25600370]
[4]
MacKenzie, G.; Maguire, J. Neurosteroids and GABAergic signaling in health and disease. Biomol. Concepts, 2013, 4(1), 29-42.
[http://dx.doi.org/10.1515/bmc-2012-0033] [PMID: 25436563]
[5]
Engin, E.; Benham, R.S.; Rudolph, U. An emerging circuit pharmacology of GABAA receptors. Trends Pharmacol. Sci., 2018, 39(8), 710-732.
[http://dx.doi.org/10.1016/j.tips.2018.04.003] [PMID: 29903580]
[6]
Olsen, R.W. GABAA receptor: Positive and negative allosteric modulators. Neuropharmacology, 2018, 136(Pt A), 10-22.
[http://dx.doi.org/10.1016/j.neuropharm.2018.01.036] [PMID: 29407219]
[7]
Solomon, V.R.; Tallapragada, V.J.; Chebib, M.; Johnston, G.A.R.; Hanrahan, J.R. GABA allosteric modulators: An overview of recent developments in non-benzodiazepine modulators. Eur. J. Med. Chem., 2019, 171, 434-461.
[http://dx.doi.org/10.1016/j.ejmech.2019.03.043] [PMID: 30928713]
[8]
Whiting, P.J. GABA-A receptor subtypes in the brain: A paradigm for CNS drug discovery? Drug Discov. Today, 2003, 8(10), 445-450.
[http://dx.doi.org/10.1016/S1359-6446(03)02703-X] [PMID: 12801796]
[9]
Belelli, D.; Harrison, N.L.; Maguire, J.; Macdonald, R.L.; Walker, M.C.; Cope, D.W. Extrasynaptic GABAA receptors: Form, pharmacology, and function. J. Neurosci., 2009, 29(41), 12757-12763.
[http://dx.doi.org/10.1523/JNEUROSCI.3340-09.2009] [PMID: 19828786]
[10]
Chua, H.C.; Chebib, M. GABAA receptors and the diversity in their structure and pharmacology. Adv. Pharmacol., 2017, 79, 1-34.
[http://dx.doi.org/10.1016/bs.apha.2017.03.003] [PMID: 28528665]
[11]
Olsen, R.W.; Sieghart, W. International Union of Pharmacology. LXX. Subtypes of gamma-aminobutyric acid(A) receptors: classification on the basis of subunit composition, pharmacology, and function. Update. Pharmacol. Rev., 2008, 60(3), 243-260.
[http://dx.doi.org/10.1124/pr.108.00505] [PMID: 18790874]
[12]
Stórustovu, S.I.; Ebert, B. Pharmacological characterization of agonists at delta-containing GABAA receptors: Functional selectivity for extrasynaptic receptors is dependent on the absence of gamma2. J. Pharmacol. Exp. Ther., 2006, 316(3), 1351-1359.
[http://dx.doi.org/10.1124/jpet.105.092403] [PMID: 16272218]
[13]
Pericić, D.; Jazvinsćak, M.; Mirković, K. [3H]Flunitrazepam binding to recombinant alpha1beta2gamma2S GABAA receptors stably expressed in HEK 293 cells. Biomed. Pharmacother., 2001, 55(4), 221-228.
[http://dx.doi.org/10.1016/S0753-3322(01)00053-1] [PMID: 11393809]
[14]
Bregestovski, P.; Waseem, T.; Mukhtarov, M. Genetically encoded optical sensors for monitoring of intracellular chloride and chloride-selective channel activity. Front. Mol. Neurosci., 2009, 2, 15.
[http://dx.doi.org/10.3389/neuro.02.015.2009] [PMID: 20057911]
[15]
Neild, T.O.; Thomas, R.C. Intracellular chloride activity and the effects of acetylcholine in snail neurones. J. Physiol., 1974, 242(2), 453-470.
[http://dx.doi.org/10.1113/jphysiol.1974.sp010717] [PMID: 4455827]
[16]
Inglefield, J.R.; Schwartz-Bloom, R.D. Confocal imaging of intracellular chloride in living brain slices: measurement of GABAA receptor activity. J. Neurosci. Methods, 1997, 75(2), 127-135.
[http://dx.doi.org/10.1016/S0165-0270(97)00054-X] [PMID: 9288644]
[17]
Marandi, N.; Konnerth, A.; Garaschuk, O. Two-photon chloride imaging in neurons of brain slices. Pflugers Arch., 2002, 445(3), 357-365.
[http://dx.doi.org/10.1007/s00424-002-0933-7] [PMID: 12466938]
[18]
Canepari, M.; Willadt, S.; Zecevic, D.; Vogt, K.E. Imaging inhibitory synaptic potentials using voltage sensitive dyes. Biophys. J., 2010, 98(9), 2032-2040.
[http://dx.doi.org/10.1016/j.bpj.2010.01.024] [PMID: 20441768]
[19]
Joesch, C.; Guevarra, E.; Parel, S.P.; Bergner, A.; Zbinden, P.; Konrad, D.; Albrecht, H. Use of FLIPR membrane potential dyes for validation of high-throughput screening with the FLIPR and microARCS technologies: identification of ion channel modulators acting on the GABA(A) receptor. J. Biomol. Screen., 2008, 13(3), 218-228.
[http://dx.doi.org/10.1177/1087057108315036] [PMID: 18270364]
[20]
Nik, A.M.; Pressly, B.; Singh, V.; Antrobus, S.; Hulsizer, S.; Rogawski, M.A.; Wulff, H.; Pessah, I.N. Rapid throughput analysis of GABAA receptor subtype modulators and blockers using DiSBAC1(3) membrane potential red dye. Mol. Pharmacol., 2017, 92(1), 88-99.
[http://dx.doi.org/10.1124/mol.117.108563] [PMID: 28428226]
[21]
Liu, J.; Chen, T.; Norris, T.; Knappenberger, K.; Huston, J.; Wood, M.; Bostwick, R. A high-throughput functional assay for characterization of gamma-aminobutyric acid(A) channel modulators using cryopreserved transiently transfected cells. Assay Drug Dev. Technol., 2008, 6(6), 781-786.
[http://dx.doi.org/10.1089/adt.2008.161] [PMID: 19090692]
[22]
Mennerick, S.; Chisari, M.; Shu, H.J.; Taylor, A.; Vasek, M.; Eisenman, L.N.; Zorumski, C.F. Diverse voltage-sensitive dyes modulate GABAA receptor function. J. Neurosci., 2010, 30(8), 2871-2879.
[http://dx.doi.org/10.1523/JNEUROSCI.5607-09.2010] [PMID: 20181584]
[23]
Lu, J.C.; Hsiao, Y.T.; Chiang, C.W.; Wang, C.T. GABAA receptor-mediated tonic depolarization in developing neural circuits. Mol. Neurobiol., 2014, 49(2), 702-723.
[http://dx.doi.org/10.1007/s12035-013-8548-x] [PMID: 24022163]
[24]
Fogaça, M.V.; Duman, R.S. Cortical GABAergic dysfunction in stress and depression: New insights for therapeutic interventions. Front. Cell. Neurosci., 2019, 13, 87.
[http://dx.doi.org/10.3389/fncel.2019.00087] [PMID: 30914923]
[25]
Sieghart, W. Allosteric modulation of GABAA receptors via multiple drug-binding sites. Adv. Pharmacol., 2015, 72, 53-96.
[http://dx.doi.org/10.1016/bs.apha.2014.10.002] [PMID: 25600367]
[26]
Johansson, T.; Norris, T.; Peilot-Sjögren, H. Yellow fluorescent protein-based assay to measure GABA(A) channel activation and allosteric modulation in CHO-K1 cells. PLoS One, 2013, 8(3), e59429.
[http://dx.doi.org/10.1371/journal.pone.0059429] [PMID: 23516634]
[27]
Gilbert, D.; Esmaeili, A.; Lynch, J.W. Optimizing the expression of recombinant alphabetagamma GABAA receptors in HEK293 cells for high-throughput screening. J. Biomol. Screen., 2009, 14(1), 86-91.
[http://dx.doi.org/10.1177/1087057108328017] [PMID: 19171924]
[28]
Zhou, X.; Desai, R.; Zhang, Y.; Stec, W.J.; Miller, K.W.; Jounaidi, Y. High-level production and purification in a functional state of an extrasynaptic gamma-aminobutyric acid type A receptor containing α4β3δ subunits. PLoS One, 2018, 13(1), e0191583.
[http://dx.doi.org/10.1371/journal.pone.0191583] [PMID: 29352320]
[29]
Elegheert, J.; Behiels, E.; Bishop, B.; Scott, S.; Woolley, R.E.; Griffiths, S.C.; Byrne, E.F.X.; Chang, V.T.; Stuart, D.I.; Jones, E.Y.; Siebold, C.; Aricescu, A.R. Lentiviral transduction of mammalian cells for fast, scalable and high-level production of soluble and membrane proteins. Nat. Protoc., 2018, 13(12), 2991-3017.
[http://dx.doi.org/10.1038/s41596-018-0075-9] [PMID: 30455477]
[30]
Muir, J.; Arancibia-Carcamo, I.L.; MacAskill, A.F.; Smith, K.R.; Griffin, L.D.; Kittler, J.T. NMDA receptors regulate GABAA receptor lateral mobility and clustering at inhibitory synapses through serine 327 on the γ2 subunit. Proc. Natl. Acad. Sci. USA, 2010, 107(38), 16679-16684.
[http://dx.doi.org/10.1073/pnas.1000589107] [PMID: 20823221]
[31]
Dostalova, Z.; Liu, A.; Zhou, X.; Farmer, S.L.; Krenzel, E.S.; Arevalo, E.; Desai, R.; Feinberg-Zadek, P.L.; Davies, P.A.; Yamodo, I.H.; Forman, S.A.; Miller, K.W. High-level expression and purification of Cys-loop ligand-gated ion channels in a tetracycline-inducible stable mammalian cell line: GABAA and serotonin receptors. Protein Sci., 2010, 19(9), 1728-1738.
[http://dx.doi.org/10.1002/pro.456] [PMID: 20662008]
[32]
Dostalova, Z.; Zhou, X.; Liu, A.; Zhang, X.; Zhang, Y.; Desai, R.; Forman, S.A.; Miller, K.W. Human α1β3γ2L gamma-aminobutyric acid type A receptors: High-level production and purification in a functional state. Protein Sci., 2014, 23(2), 157-166.
[http://dx.doi.org/10.1002/pro.2401] [PMID: 24288268]
[33]
Cao, Z.; Zou, X.; Cui, Y.; Hulsizer, S.; Lein, P.J.; Wulff, H.; Pessah, I.N. Rapid throughput analysis demonstrates that chemicals with distinct seizurogenic mechanisms differentially alter Ca2+ dynamics in networks formed by hippocampal neurons in culture. Mol. Pharmacol., 2015, 87(4), 595-605.
[http://dx.doi.org/10.1124/mol.114.096701] [PMID: 25583085]
[34]
Inoue, M.; Harada, K.; Nakamura, J.; Matsuoka, H.; Fau-Matsuoka, H.; Matsuoka, H. Regulation of α3-containing GABAA receptors in guinea-pig adrenal medullary cells by adrenal steroids. Neuroscience, 2013, 253, 245-255.
[http://dx.doi.org/10.1016/j.neuroscience.2013.08.046] [PMID: 24012744]
[35]
Fu, Z.; Vicini, S. Neuroligin-2 accelerates GABAergic synapse maturation in cerebellar granule cells. Mol. Cell. Neurosci., 2009, 42(1), 45-55.
[http://dx.doi.org/10.1016/j.mcn.2009.05.004] [PMID: 19463950]
[36]
Ng, C.C.; Duke, R.K.; Hinton, T.; Johnston, G.A.R. Effects of bilobalide, ginkgolide B and picrotoxinin on GABAA receptor modulation by structurally diverse positive modulators. Eur. J. Pharmacol., 2017, 806, 83-90.
[http://dx.doi.org/10.1016/j.ejphar.2017.04.019] [PMID: 28416372]
[37]
Hansen, S.L.; Ebert, B.; Fjalland, B.; Kristiansen, U. Effects of GABA(A) receptor partial agonists in primary cultures of cerebellar granule neurons and cerebral cortical neurons reflect different receptor subunit compositions. Br. J. Pharmacol., 2001, 133(4), 539-549.
[http://dx.doi.org/10.1038/sj.bjp.0704121] [PMID: 11399671]
[38]
Ghansah, E.; Weiss, D.S. Benzodiazepines do not modulate desensitization of recombinant alpha1beta2gamma2 GABA(A) receptors. Neuroreport, 1999, 10(4), 817-821.
[http://dx.doi.org/10.1097/00001756-199903170-00028] [PMID: 10208554]
[39]
Huang, Q.T.; Sheng, C.W.; Jiang, J.; Tang, T.; Jia, Z.Q.; Han, Z.J.; Zhao, C.Q. Interaction of insecticides with heteromeric GABA-gated chloride channels from zebrafish Danio rerio (Hamilton). J. Hazard. Mater., 2019, 366, 643-650.
[http://dx.doi.org/10.1016/j.jhazmat.2018.11.085] [PMID: 30580138]
[40]
Zhang, J.; Xue, F.; Chang, Y. Structural determinants for antagonist pharmacology that distinguish the rho1 GABAC receptor from GABAA receptors. Mol. Pharmacol., 2008, 74(4), 941-951.
[http://dx.doi.org/10.1124/mol.108.048710] [PMID: 18599601]
[41]
Huang, L.; Ding, J.; Li, M.; Hou, Z.; Geng, Y.; Li, X.; Yu, H. Discovery of [1,2,4]-triazolo [1,5-a]pyrimidine-7(4H)-one derivatives as positive modulators of GABAA1 receptor with potent anticonvulsant activity and low toxicity. Eur. J. Med. Chem., 2020, 185, 111824.
[http://dx.doi.org/10.1016/j.ejmech.2019.111824] [PMID: 31708184]
[42]
Wu, J.; Hou, Z.; Wang, Y.; Chen, L.; Lian, C.; Meng, Q.; Zhang, C.; Li, X.; Huang, L.; Yu, H. Discovery of 7-alkyloxy- [1,2,4] triazolo[1,5-a] pyrimidine derivatives as selective positive modulators of GABAA1 and GABAA4 receptors with potent antiepileptic activity. Bioorg. Chem., 2022, 119, 105565.
[http://dx.doi.org/10.1016/j.bioorg.2021.105565] [PMID: 34929519]

Rights & Permissions Print Cite
Article Metrics
36
3
© 2024 Bentham Science Publishers | Privacy Policy