title: Implementation of a fluorescence-based screening assay identifies histamine H3 receptor antagonists clobenpropit and iodophenpropit as subunit-selective N-methyl-D-aspartate receptor antagonists.
abstract: N-Methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels that mediate a slow, Ca(2+)-permeable component of excitatory synaptic transmission in the central nervous system and play a pivotal role in synaptic plasticity, neuronal development, and several neurological diseases. We describe a fluorescence-based assay that measures NMDA receptor-mediated changes in intracellular calcium in a BHK-21 cell line stably expressing NMDA receptor NR2D with NR1 under the control of a tetracycline-inducible promoter (Tet-On). The assay selectively identifies allosteric modulators by using supramaximal concentrations of glutamate and glycine to minimize detection of competitive antagonists. The assay is validated by successfully identifying known noncompetitive, but not competitive NMDA receptor antagonists among 1800 screened compounds from two small focused libraries, including the commercially available library of pharmacologically active compounds. Hits from the primary screen are validated through a secondary screen that used two-electrode voltage-clamp recordings on recombinant NMDA receptors expressed in Xenopus laevis oocytes. This strategy identified several novel modulators of NMDA receptor function, including the histamine H3 receptor antagonists clobenpropit and iodophenpropit, as well as the vanilloid receptor transient receptor potential cation channel, subfamily V, member 1 (TRPV1) antagonist capsazepine. These compounds are noncompetitive antagonists and the histamine H3 receptor ligand showed submicromolar potency at NR1/NR2B NMDA receptors, which raises the possibility that compounds can be developed that act with high potency on both glutamate and histamine receptor systems simultaneously. Furthermore, it is possible that some actions attributed to histamine H3 receptor inhibition in vivo may also involve NMDA receptor antagonism.
authors.affiliation: Department of Pharmacology, Emory University School of Medicine, Rollins Research Center, Atlanta, Georgia 30322, USA.
authors.forename: Kasper B
authors.initials: KB
authors.lastname: Hansen
authors.forename: Praseeda
authors.initials: P
authors.lastname: Mullasseril
authors.forename: Sara
authors.initials: S
authors.lastname: Dawit
authors.forename: Natalie L
authors.initials: NL
authors.lastname: Kurtkaya
authors.forename: Hongjie
authors.initials: H
authors.lastname: Yuan
authors.forename: Katie M
authors.initials: KM
authors.lastname: Vance
authors.forename: Anna G
authors.initials: AG
authors.lastname: Orr
authors.forename: Trine
authors.initials: T
authors.lastname: Kvist
authors.forename: Kevin K
authors.initials: KK
authors.lastname: Ogden
authors.forename: Phuong
authors.initials: P
authors.lastname: Le
authors.forename: Kimberly M
authors.initials: KM
authors.lastname: Vellano
authors.forename: Iestyn
authors.initials: I
authors.lastname: Lewis
authors.forename: Serdar
authors.initials: S
authors.lastname: Kurtkaya
authors.forename: Yuhong
authors.initials: Y
authors.lastname: Du
authors.forename: Min
authors.initials: M
authors.lastname: Qui
authors.forename: T J
authors.initials: TJ
authors.lastname: Murphy
authors.forename: James P
authors.initials: JP
authors.lastname: Snyder
authors.forename: Hans
authors.initials: H
authors.lastname: Bräuner-Osborne
authors.forename: Stephen F
authors.initials: SF
authors.lastname: Traynelis
mesh_terms: D000814:Aniline Compounds; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D004353:Drug Evaluation, Preclinical; D004594:Electrophysiology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005456:Fluorescent Dyes; D054828:Histamine H3 Antagonists / Q000494:pharmacology*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D007550:Isothiuronium / Q000031:analogs & derivatives* / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D009865:Oocytes / Q000187:drug effects; D018408:Patch-Clamp Techniques; D010880:Piperidines / Q000494:pharmacology; D011869:Radioligand Assay; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics; D013329:Structure-Activity Relationship; D013890:Thiourea / Q000031:analogs & derivatives* / Q000494:pharmacology; D014966:Xanthenes; D014982:Xenopus laevis
chemical_list: D000814:Aniline Compounds; D018691:Excitatory Amino Acid Antagonists; C409648:Fluo 4; D005456:Fluorescent Dyes; D054828:Histamine H3 Antagonists; D007093:Imidazoles; C409466:NR1 NMDA receptor; C121001:NR2B NMDA receptor; D010880:Piperidines; D016194:Receptors, N-Methyl-D-Aspartate; D014966:Xanthenes; D007550:Isothiuronium; C077791:iodophenpropit; D013890:Thiourea; C010739:ifenprodil; C086108:clobenpropit