title: Kinetic characterization of novel NR2B antagonists using fluorescence detection of calcium flux.
abstract: To facilitate the discovery of novel N-methyl-d-aspartate (NMDA) receptor antagonists, we have developed a high-throughput functional assay based on fluorescence detection of free intracellular calcium concentrations. Mouse fibroblast L(tk-) cells expressing human NR1a/NR2B NMDA receptors were plated in 96-well plates and loaded with fluorescence calcium indicator fluo-3 AM. NR2B antagonists were added after stimulation of NMDA receptors with 10 microM glutamate and 10 microM glycine. Changes in fluorescence after the addition of the antagonists were fitted by a single exponential equation providing k(obs). The concentration dependence of k(obs) was linear for all NR2B antagonists at concentrations where k(obs) < 0.2 s(-1). The values of k(obs) for six structurally distinct NR2B antagonists were in the range of 1.1 to 7.5 x 10(5) M(-1)s(-1). These values were several orders of magnitude slower than that obtained for diffusion limited Mg(2+) channel block. The rate constants k(off) provided the values of t(1/2) for dissociation of NR2B antagonists in the range of 1.8 min for ifenprodil to 240 min for the slowest novel antagonist. The IC(50) values obtained from the end-point fluorescence measurements agree with K(d) values calculated from kinetic measurements. All kinetic constants, obtained using our fluorescence method, correlate well with data measured by voltage clamp.
authors.affiliation: Department of Neurology, Merck Research Laboratories, WP26A-2000 Sumneytown Pike, West Point, PA 19454, USA. bohumil_bednar@merck.com
authors.forename: Bohumil
authors.initials: B
authors.lastname: Bednar
authors.forename: Michael E
authors.initials: ME
authors.lastname: Cunningham
authors.forename: Laszlo
authors.initials: L
authors.lastname: Kiss
authors.forename: Gong
authors.initials: G
authors.lastname: Cheng
authors.forename: John A
authors.initials: JA
authors.lastname: McCauley
authors.forename: Nigel J
authors.initials: NJ
authors.lastname: Liverton
authors.forename: Kenneth S
authors.initials: KS
authors.lastname: Koblan
mesh_terms: D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line / Q000187:drug effects / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D018691:Excitatory Amino Acid Antagonists / Q000737:chemistry / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051379:Mice; D008959:Models, Neurological; D018408:Patch-Clamp Techniques / Q000379:methods*; D010880:Piperidines / Q000494:pharmacology; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors; D014162:Transfection / Q000379:methods; D014966:Xanthenes / Q000378:metabolism
chemical_list: D000814:Aniline Compounds; D018691:Excitatory Amino Acid Antagonists; D005456:Fluorescent Dyes; C121001:NR2B NMDA receptor; D010880:Piperidines; D016194:Receptors, N-Methyl-D-Aspartate; D014966:Xanthenes; C059715:Fluo-3; C010739:ifenprodil; D002118:Calcium