Abstract
NR1/NR2A is a subtype of N-methyl-d-aspartate receptors (NMDARs), which are glutamate and glycine-gated Ca2+-permeable channels highly expressed in the central nervous system. A high-throughput screening (HTS) campaign using human osteosarcoma (U-2 OS) cells transiently transduced with NR1/NR2A NMDAR subunits, tested in a specifically designed fluorometric imaging plate reader (FLIPR)/Ca2+ assay, identified sulfonamide derivative series, exemplified by 3-chloro-4-fluoro-N-[(4-{[2-(phenylcarbonyl)hydrazino]carbonyl}phenyl)methyl]benzenesulfonamide (compound 1) and thiodiazole derivative N-(cyclohexylmethyl)-2-({5-[(phenylmethyl)amino]-1,3,4-thiadiazol-2-yl}thio)acetamide (compound 13) as novel NR1/NR2A receptor antagonists. Compounds 1 and 13 displayed submicromolar and micromolar potency at NR1/NR2A receptor, respectively, although they did not show activity at NR2B-containing receptor up to 50 μM concentration. Addition of 1 mM glycine, but not 1 mM l-glutamate, was able to surmount compound 1 and 13 inhibitory effects in FLIPR NR1/NR2A assay. However, compounds 1 and 13 displaced a glutamate site antagonist [3H]d,l-(E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid ([3H]CGP 39653) to a greater extent than the glycine site antagonist [3H]3-[(E)-2-carboxy-2-phenylethenyl]-4,6-dichloro-1H-indole-2-carboxylic acid ([3H]MDL 105,519), in rat brain cortex binding assay. Results of FLIPR cell-based, electrophysiological, and biochemical binding assays suggest that compounds 1 and 13 are the prototypes of novel classes of NMDAR ligands, which to the best of our knowledge are the first selective antagonists at NR1/NR2A over NR1/NR2B receptor, and might constitute useful tools able to elucidate the relative role of the NR2A subunit in physiological and pathological conditions.
Footnotes
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.110.172544.
↵
The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.-
ABBREVIATIONS:
- NMDAR
- N-methyl-d-aspartate receptor
- ABD
- agonist binding domain
- AM
- acetoxymethyl
- CGP 39653
- d,l-(E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid
- CP-101,606
- traxoprodil
- CRC
- concentration response curve
- compound 1
- 3-chloro-4-fluoro-N-[(4-{[2-(phenylcarbonyl)hydrazino]carbonyl}phenyl)methyl]benzenesulfonamide
- compound 2
- 3-chloro-N-[4-({2-[(2,5-dimethylfuran-3-yl)carbonyl]hydrazinyl}carbonyl)benzyl]-4-fluorobenzenesulfonamide
- compound 3
- 3-chloro-4-fluoro-N-(4-{[2-(pyridin-3-ylcarbonyl)hydrazinyl]carbonyl}benzyl)benzenesulfonamide
- compound 4
- 3-chloro-N-(4-{[2-(cyclohexylcarbonyl)hydrazino]carbonyl}benzyl)-4-fluorobenzenesulfonamide
- compound 5
- N-(4-{[2-(2-fluorobenzoyl)hydrazinyl]carbonyl}benzyl)-N,4-dimethylbenzenesulfonamide
- compound 6
- 4-({[(4-fluorophenyl)sulfonyl]amino}methyl)-N-(pyridin-3-ylmethyl)benzamide
- compound 7
- 4-({[(3-chloro-4-fluorophenyl)sulfonyl]amino}methyl)-N-(tetrahydrofuran-2-ylmethyl)benzamide
- compound 8
- 4-({[(3-chloro-4-fluorophenyl)sulfonyl]amino}methyl)-N-(pyridin-4-ylmethyl)benzamide
- compound 9
- N-(4-chlorobenzyl)-4-({[(4-fluorophenyl)sulfonyl]amino}methyl)benzamide; compound 10,4-({[(3-chloro-4-fluorophenyl)sulfonyl]amino}methyl)-N-(pyridin-2-ylmethyl)benzamide
- compound 11
- N-benzyl-4-({[(4-fluorophenyl)sulfonyl]amino}methyl)-N-methylbenzamide
- compound 12
- 4-[benzenesulfonyl(methyl)amino]-N-(pyridin-3-ylmethyl)benzamide
- compound 13
- N-(cyclohexylmethyl)-2-({5-[(phenylmethyl)amino]-1,3,4-thiadiazol-2-yl}thio)acetamide
- DMSO
- dimethyl sulfoxide
- FLIPR
- fluorometric imaging plate reader
- GV196771A
- sodium 4,6-dichloro-3-[(E)-(2-oxo-1-phenyl-3-pyrrolidinylidene)methyl]-1H-indole-2-carboxylate
- GFP
- green fluorescent protein
- HBSSH
- Hanks' balanced salt solution supplemented with HEPES
- HEK
- human embryonic kidney
- HTS
- high-throughput screening
- LE
- ligand efficiency
- LLE
- ligand lipophilicity efficiency
- MDL 105,519
- 3-[(E)-2-carboxy-2-phenylethenyl]-4,6-dichloro-1H-indole-2-carboxylic acid
- (+)-MK-801
- dizocilpine maleate
- NMDA
- N-methyl-d-aspartate
- NTD
- N-terminal domain
- NVP-AAM077
- [(R)-{[(1S)-1-(4-bromophenyl)ethyl]amino}(2,3-dioxo-1,2,3,4-tetrahydro-5-quinoxalinyl)methyl]phosphonic acid
- EAA-090
- perzinfotel
- Ro 25-6981
- 4-{(1R,2S)-1-hydroxy-2-methyl-3-[4-(phenylmethyl)-1-piperidinyl]propyl}phenol
- TCP
- 1-(1-(2-thienyl)cyclohexyl)piperidine.
- Received July 15, 2010.
- Accepted August 24, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|
