Co-reporter:Nasiara Karim;Petrine Wellendorph;Nathan Absalom;Graham A. R. Johnston
Amino Acids 2013 Volume 44( Issue 4) pp:1139-1149
Publication Date(Web):2013 April
DOI:10.1007/s00726-012-1456-y
GABAA receptors are members of the ligand-gated ion channel superfamily that mediate inhibitory neurotransmission in the central nervous system. They are thought to be composed of 2 alpha (α), 2 beta (β) subunits and one other such as a gamma (γ) or delta (δ) subunit. The potency of GABA is influenced by the subunit composition. However, there are no reported systematic studies that evaluate GABA potency on a comprehensive number of subunit combinations expressed in Xenopus oocytes, despite the wide use of this heterologous expression system in structure–function studies and drug discovery. Thus, the aim of this study was to conduct a systematic characterization of the potency of GABA at 43 human recombinant GABAA receptor combinations expressed in Xenopus oocytes using the two-electrode voltage clamp technique. The results show that the α-subunits and to a lesser extent, the β-subunits influence GABA potency. Of the binary and ternary combinations with and without the γ2L subunit, the α6/γ2L-containing receptors were the most sensitive to GABA, while the β2- or β3-subunit conferred higher sensitivity to GABA than receptors containing the β1-subunit with the exception of the α2β1γ2L and α6β1γ2L subtypes. Of the δ-subunit containing GABAA receptors, α4/δ-containing GABAA receptors displayed highest GABA sensitivity, with mid-nanomolar concentrations activating α4β1δ and α4β3δ receptors. At α4β2δ, GABA had low micromolar activity.
Co-reporter:Navnath Gavande, Izumi Yamamoto, Noeris K. Salam, Tu-Hoa Ai, Peter M. Burden, Graham A. R. Johnston, Jane R. Hanrahan, and Mary Chebib
ACS Medicinal Chemistry Letters 2011 Volume 2(Issue 1) pp:11
Publication Date(Web):October 19, 2010
DOI:10.1021/ml1001344
Understanding the role of GABAC receptors in the central nervous system is limited due to a lack of specific ligands. Novel γ-aminobutyric acid (GABA) analogues based on 3-(aminomethyl)-1-oxo-1-hydroxy-phospholane 17 and 3-(guanido)-1-oxo-1-hydroxy-phospholane 19 were investigated to obtain selective GABAC receptor antagonists. A compound of high potency (19, KB = 10 μM) and selectivity (greater than 100 times at ρ1 GABAC receptors as compared to α1β2γ2L GABAA and GABAB(1b,2) receptors) was obtained. The cyclic phosphinic acids (17 and 19) are novel lead agents for developing into more potent and selective GABAC receptor antagonists with increased lipophilicity for future in vivo studies.Keywords (keywords): cyclic phosphinic acids; GABA receptors; ligand-gated ion channels; two-electrode voltage clamp; γ-Aminobutyric acid; ρ1 GABAC homology model
Co-reporter:Navnath Gave;Nasiara Karim; Graham A. R. Johnston; Jane R. Hanrahan; Mary Chebib
ChemMedChem 2011 Volume 6( Issue 8) pp:1340-1346
Publication Date(Web):
DOI:10.1002/cmdc.201100120
Co-reporter:Navnath Gave;Nasiara Karim; Graham A. R. Johnston; Jane R. Hanrahan; Mary Chebib
ChemMedChem 2011 Volume 6( Issue 8) pp:
Publication Date(Web):
DOI:10.1002/cmdc.201190032
Co-reporter:Navnath Gavande, Graham A. R. Johnston, Jane R. Hanrahan and Mary Chebib
Organic & Biomolecular Chemistry 2010 vol. 8(Issue 18) pp:4131-4136
Publication Date(Web):26 Jul 2010
DOI:10.1039/C0OB00004C
Microwave-enhanced, highly efficient protocols for the synthesis of synthetically and biologically important 2,3,6-trisubstituted pyridazine architectures have been developed by sequential amination/Suzuki coupling/alkylation reactions. This powerful strategy is an economical and highly chemoselective protocol for the synthesis of diversified pyridazines. The total synthesis of gabazine (SR-95531) has been achieved using a versatile strategy in four steps and 73% overall yield.
Co-reporter:Mary Chebib;Navnath Gavande;Kit Yee Wong;Anna Park
Neurochemical Research 2009 Volume 34( Issue 10) pp:1704-1711
Publication Date(Web):2009 October
DOI:10.1007/s11064-009-9968-x
GABAC receptors play a role in myopia, memory-related disorders and circadian rhythms signifying a need to develop potent and selective agents for this class of receptors. Guanidino analogs related to glycine, β-alanine and taurine were evaluated at human ρ1GABAC receptors expressed in Xenopus oocytes using 2-electrode voltage clamp methods. Of the 12 analogs tested, 8 analogs were active as antagonists and the remaining were inactive. (S)-2-Guanidinopropionic acid (IC50 = 2.2 μM) and guanidinoacetic acid (IC50 = 5.4 μM; KB = 7.75 μM [pKB = 5.11 ± 0.06]) were the most potent being competitive antagonists at this receptor. In contrast, the β-alanine and GABA guanidino analogs showed reduced activity, indicating the distance between the carboxyl carbon and terminal nitrogen of the guanidino group is critical for activity. Substituting the C2-position of guanidinoacetic acid with various alkyl groups reduced activity indicating that steric effects may impact on activity. The results of this study contribute to the structure–activity-relationship profile required in developing novel therapeutic agents.
Co-reporter:Heba Abdel-Halim;Jane R. Hanrahan;David E. Hibbs;Graham A. R. Johnston
Chemical Biology & Drug Design 2008 Volume 71( Issue 4) pp:306-327
Publication Date(Web):
DOI:10.1111/j.1747-0285.2008.00642.x
We modelled the N-terminal ligand-binding domain of the ρ1 GABAC receptor based on the Lymnaea stagnalis acetylcholine-binding protein (L-AChBP) crystal structure using comparative modelling and validated using flexible docking guided by known mutagenesis studies. A range of known ρ1 GABAC receptor ligands comprising seven full agonists, 10 partial agonists, 43 antagonists and 12 inactive molecules were used to evaluate and validate the models. Of the 50 models identified, six models that allowed flexible ligand docking in accordance with the experimental data were selected and used to study detailed receptor-ligand interactions. The most refined model to accommodate all known active ligands featured a cavity comprising of a volume of 488 Å3. A detailed analysis of the interaction between the ρ1 GABAC receptor model and the docked ligands revealed possible H-bonds and cation-π interactions between the different ligands and binding site residues. Based on quantum mechanical/molecular mechanical (QM/MM) calculations, the model showed distinctive conformations of loop C that provided a molecular basis for agonist and antagonist actions. Agonists elicit loop C closure, while a more open loop C was observed upon antagonist binding. The model differentiates the role for key residues known to be involved in either binding and/or gating.
Co-reporter:Deborah L. Crittenden, Anna Park, Jian Qiu, Richard B. Silverman, Rujee K. Duke, Graham A.R. Johnston, Meredith J.T. Jordan, Mary Chebib
Bioorganic & Medicinal Chemistry 2006 Volume 14(Issue 2) pp:447-455
Publication Date(Web):15 January 2006
DOI:10.1016/j.bmc.2005.08.037
The effects of the enantiomers of a number of flexible and cis-constrained GABA analogues were tested on GABAC receptors expressed in Xenopus laevis oocytes using two-electrode voltage-clamp electrophysiology. (1S,2R)-cis-2-Aminomethylcyclopropane-1-carboxylic acid ((+)-CAMP), a potent and full agonist at the ρ1 (EC50 ≈ 40 μM, Imax ≈ 100%) and ρ 2 (EC50 ≈ 17 μM, Imax ≈ 100%) receptor subtypes, was found to be a potent partial agonist at ρ3 (EC50 ≈ 28 μM, Imax ≈ 70%). (1R,2S)-cis-2-Aminomethylcyclopropane-1-carboxylic acid ((−)-CAMP), a weak antagonist at human ρ1 (IC50 ≈ 890 μM) and ρ2 (IC50 ≈ 400 μM) receptor subtypes, was also found to be a moderately potent antagonist at rat ρ3 (IC50 ≈ 180 μM). Similarly, (1R,4S)-4-aminocyclopent-2-ene-1-carboxylic acid ((+)-ACPECA) was a full agonist at ρ1 (EC50 ≈ 135 μM, Imax ≈ 100%) and ρ2 (EC50 ≈ 60 μM, Imax ≈ 100%), but only a partial agonist at ρ3 (EC50 ≈ 112 μM, Imax ≈ 37 %), while (1S,4R)-4-aminocyclopent-2-ene-1-carboxylic acid ((−)-ACPECA) was a weak antagonist at all three receptor subtypes (IC50 >> 300 μM). 4-Amino-(S)-2-methylbutanoic acid ((S)-2MeGABA) and 4-amino-(R)-2-methylbutanoic acid ((R)-2MeGABA) followed the same trend, with (S)-2MeGABA acting as a full agonist at the ρ1 (EC50 ≈ 65 μM, Imax ≈ 100%), and ρ2 (EC50 ≈ 20 μM, Imax ≈ 100%) receptor subtypes, and a partial agonist at ρ3 (EC50 ≈ 25 μM, Imax ≈ 90%). (R)-2MeGABA, however, was a moderately potent antagonist at all three receptor subtypes (IC50 ≈ 16 μM at ρ1, 125 μM at ρ2 and 35 μM at ρ3). On the basis of these expanded biological activity data and the solution-phase molecular structures obtained at the MP2/6-31+G* level of ab initio theory, a rationale is proposed for the genesis of this stereoselectivity effect.The effects of the enantiomers of a number of flexible and cis-constrained GABA analogues are tested on GABAC receptors expressed in Xenopus Laevis oocytes using two-electrode voltage-clamp electrophysiology. Different enantiomers are found to have opposite biological activities, with the (S) and (+) enantiomers possessing antagonist activity and the (R) and (−) enantiomers possessing antagonist activity. A novel stereoselective binding mechanism is proposed to explain this effect.
Co-reporter:Jimmy Vien;Rujee K Duke;Kenneth N Mewett;Graham A R Johnston;Ryuzo Shingai
British Journal of Pharmacology 2002 Volume 135(Issue 4) pp:
Publication Date(Web):2 FEB 2009
DOI:10.1038/sj.bjp.0704432
This study investigated the effects of a number of GABA analogues on rat ρ3 GABAC receptors expressed in Xenopus oocytes using 2-electrode voltage clamp methods.
The potency order of agonists was muscimol (EC50=1.9±0.1 μM) (+)-trans-3-aminocyclopentanecarboxylic acids ((+)-TACP; EC50=2.7±0.9 μM) trans-4-aminocrotonic acid (TACA; EC50=3.8±0.3 μM) GABA (EC50=4.0±0.3 μM) > thiomuscimol (EC50=24.8±2.6 μM) > (±)-cis-2-aminomethylcyclopropane-carboxylic acid ((±)-CAMP; EC50=52.6±8.7 μM) > cis-4-aminocrotonic acid (CACA; EC50=139.4±5.2 μM).
The potency order of antagonists was (±)-trans-2-aminomethylcyclopropanecarboxylic acid ((±)-TAMP; KB=4.8±1.8 μM) (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA; KB=4.8±0.8 μM) > (piperidin-4-yl)methylphosphinic acid (P4MPA; KB=10.2±2.3 μM) 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP; KB=10.2±0.3 μM) imidazole-4-acetic acid (I4AA; KB=12.6±2.7 μM) > 3-aminopropylphosphonic acid (3-APA; KB=35.8±13.5 μM).
trans-4-Amino-2-methylbut-2-enoic acid (2-MeTACA; 300 μM) had no effect as an agonist or an antagonist indicating that the C2 methyl substituent is sterically interacting with the ligand-binding site of rat ρ3 GABAC receptors.
2-MeTACA affects ρ1 and ρ2 but not ρ3 GABAC receptors. In contrast, (±)-TAMP is a partial agonist at ρ1 and ρ2 GABAC receptors, while at rat ρ3 GABAC receptors it is an antagonist. Thus, 2-MeTACA and (±)-TAMP could be important pharmacological tools because they may functionally differentiate between ρ1, ρ2 and ρ3 GABAC receptors in vitro.
This study investigated the effects of a number of GABA analogues on rat ρ3 GABAC receptors expressed in Xenopus oocytes using 2-electrode voltage clamp methods.
The potency order of agonists was muscimol (EC50=1.9±0.1 μM) (+)-trans-3-aminocyclopentanecarboxylic acids ((+)-TACP; EC50=2.7±0.9 μM) trans-4-aminocrotonic acid (TACA; EC50=3.8±0.3 μM) GABA (EC50=4.0±0.3 μM) > thiomuscimol (EC50=24.8±2.6 μM) > (±)-cis-2-aminomethylcyclopropane-carboxylic acid ((±)-CAMP; EC50=52.6±8.7 μM) > cis-4-aminocrotonic acid (CACA; EC50=139.4±5.2 μM).
The potency order of antagonists was (±)-trans-2-aminomethylcyclopropanecarboxylic acid ((±)-TAMP; KB=4.8±1.8 μM) (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA; KB=4.8±0.8 μM) > (piperidin-4-yl)methylphosphinic acid (P4MPA; KB=10.2±2.3 μM) 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP; KB=10.2±0.3 μM) imidazole-4-acetic acid (I4AA; KB=12.6±2.7 μM) > 3-aminopropylphosphonic acid (3-APA; KB=35.8±13.5 μM).
trans-4-Amino-2-methylbut-2-enoic acid (2-MeTACA; 300 μM) had no effect as an agonist or an antagonist indicating that the C2 methyl substituent is sterically interacting with the ligand-binding site of rat ρ3 GABAC receptors.
2-MeTACA affects ρ1 and ρ2 but not ρ3 GABAC receptors. In contrast, (±)-TAMP is a partial agonist at ρ1 and ρ2 GABAC receptors, while at rat ρ3 GABAC receptors it is an antagonist. Thus, 2-MeTACA and (±)-TAMP could be important pharmacological tools because they may functionally differentiate between ρ1, ρ2 and ρ3 GABAC receptors in vitro.
British Journal of Pharmacology (2002) 135, 883–890; doi:10.1038/sj.bjp.0704432
Co-reporter:Navnath Gavande, Graham A. R. Johnston, Jane R. Hanrahan and Mary Chebib
Organic & Biomolecular Chemistry 2010 - vol. 8(Issue 18) pp:NaN4136-4136
Publication Date(Web):2010/07/26
DOI:10.1039/C0OB00004C
Microwave-enhanced, highly efficient protocols for the synthesis of synthetically and biologically important 2,3,6-trisubstituted pyridazine architectures have been developed by sequential amination/Suzuki coupling/alkylation reactions. This powerful strategy is an economical and highly chemoselective protocol for the synthesis of diversified pyridazines. The total synthesis of gabazine (SR-95531) has been achieved using a versatile strategy in four steps and 73% overall yield.
