Application of MS Transport Assays to the Four Human γ‐Aminobutyric Acid Transporters

Abstract

γ-Aminobutyric acid (GABA) transporters (GATs) are promising drug targets for various diseases associated with imbalances in GABAergic neurotransmission. For the development of new drugs or pharmacological tools addressing GATs, screening techniques to identify new inhibitors and to characterize their potency at each GAT subtype are indispensable. By now, the technique by far dominating is based on radiolabeled GABA. We recently described "MS Transport Assays" for hGAT-1 by employing ((2) H6 )GABA as the substrate. In the present study, we applied this approach to all four human GAT subtypes and determined the KM values for GAT-mediated transport of ((2) H6 )GABA at each subtype. Furthermore, a comprehensive set of GAT inhibitors reflecting the whole range of potency and subtype selectivity known so far was evaluated for their potency. The comparison of pIC50 values obtained in conventional [(3) H]GABA uptake assays with those obtained in MS Transport Assays indicated the reliability of the latter. The MS Transport Assays enable a throughput similar to that of conventional radiometric transport assays performed in a 96-well format but avoid the use of radiolabeled substrates.

Development of Highly Potent GAT1 Inhibitors: Synthesis of Nipecotic Acid Derivatives by Suzuki–Miyaura Cross‐Coupling Reactions

“…The respective compound, 22 e (Table , entry 6), achieved a p K i value of 8.05±0.13 and a pIC 50 value of 7.28±0.08. Finally, the ( R )‐enantiomer (( R )‐ 22 e ; Table , entry 7) (DDPM‐2349) displayed a p K i value of 8.33±0.06 and a pIC 50 of 7.43±0.10, thus improving the binding affinity of ( E )‐ 9 by more than one and the inhibitory potency by half a log unit. In addition, compound ( R )‐ 22 e displayed subtype selectivity in inhibitory potency in favor of mGAT1, as compared to mGAT2–mGAT4, of about three log units.…”

“…[e] Compound displaying fully saturated C4 linker. ChemMedChem 2016, 11,519 -538 www.chemmedchem.org enantiomer ((R)-22 e;T able 2, entry 7) (DDPM-2349 [17] )d isplayed ap K i value of 8.33 AE 0.06 and ap IC 50 of 7.43 AE 0.10, thus improving the bindinga ffinity of (E)-9 by more than one and the inhibitory potencyb yh alf al og unit. In addition, compound (R)-22 e displayed subtype selectivity in inhibitory potency in favor of mGAT1, as compared to mGAT2-mGAT4, of about three log units.…”

“…Envisaging compounds bearing an aromatic domain at only one position of the vinyl moiety, we synthesized a small series of biphenylic nipecotic acid derivatives, with the two aryl groups of the biphenyl moiety emulating a geminal substitution, as in 3 . This motif has not been fully investigated to the best of our knowledge so far . A preliminary evaluation of the scaffold led to compound ( E )‐ 9 , which we chose as our hit compound after testing its inhibitory activity toward mGAT1 (pIC 50 =6.79±0.16).…”

“…Envisaging compounds bearing an aromatic domain at only one positiono ft he vinyl moiety,w es ynthesized as mall series of biphenylic nipecotic acid derivatives, with the two aryl groups of the biphenyl moiety emulating ag eminal substitution, as in 3.T his motif hasn ot been fully investigated to the best of our knowledge so far. [15][16][17] Ap reliminarye valuation of the scaffold led to compound (E)-9,w hich we chose as our hit compound after testing its inhibitory activity toward mGAT1 (pIC 50 = 6.79 AE 0.16). With this active hitinh and, we focusedo n R 1 as an anchorp oint for an ortho-biphenylm oiety and began to probe the SAR space around the distal aromatic ring of this group, assisted by in silico docking studies.…”

“…Reagents and conditions:a)H 2 ,10% Pd/C, 5bar,RT, 4h,8 9%.Scheme6.Synthesis of mGATinhibitors 30 a,c from building block 28. Reagents and conditions:a)11,[P(Me) 3 CH 2 CN] + I À ,DIPEA, MeCN,reflux,2 .5 h, 92 %; b) RBr (14 a,c), PdCl 2 (dppf)·CH 2 Cl 2 (5 %), K 2 CO 3 ,d ioxane/H 2 O(5:1), 60 8C, 7-8 h; c) NaOH, EtOH, RT,[9][10][11][12][13][14][15][16][17][18][19][20] Scheme7.Synthesis of (Z)-9. Reagents and conditions:a)(PPh 3 ) 2 PdCl 2 (0.02equiv), tributyltin hydride( 1.1 equiv), THF,RT, 1h,40%;b )TFA/H 2 O( 50 %), RT,1h, 69 %; c) NaOH( 12 m), RT,6h, 100 %.…”