Implementation of a fluorescence-based screening assay identifies histamine H3 receptor antagonists clobenpropit and iodophenpropit as subunit-selective N-methyl-D-aspartate receptor antagonists.

TitleImplementation of a fluorescence-based screening assay identifies histamine H3 receptor antagonists clobenpropit and iodophenpropit as subunit-selective N-methyl-D-aspartate receptor antagonists.
Publication TypeJournal Article
Year of Publication2010
AuthorsHansen KB, Mullasseril P, Dawit S, Kurtkaya NL, Yuan H, Vance KM, Orr AG, Kvist T, Ogden KK, Le P, Vellano KM, Lewis I, Kurtkaya S, Du Y, Qui M, Murphy TJ, Snyder JP, Bräuner-Osborne H, Traynelis SF
JournalJ Pharmacol Exp Ther
Volume333
Issue3
Pagination650-62
Date Published2010 Jun
ISSN1521-0103
KeywordsAniline Compounds, Animals, Cell Line, Cricetinae, Drug Evaluation, Preclinical, Electrophysiology, Excitatory Amino Acid Antagonists, Fluorescent Dyes, Histamine H3 Antagonists, Humans, Imidazoles, Isothiuronium, Microscopy, Fluorescence, Oocytes, Patch-Clamp Techniques, Piperidines, Radioligand Assay, Receptors, N-Methyl-D-Aspartate, Structure-Activity Relationship, Thiourea, Xanthenes, Xenopus laevis
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.

DOI10.1124/jpet.110.166256
Alternate JournalJ. Pharmacol. Exp. Ther.
PubMed ID20197375
PubMed Central IDPMC2879924
Grant ListNS036654 / NS / NINDS NIH HHS / United States
U54 HG003918 / HG / NHGRI NIH HHS / United States
R37 NS036654 / NS / NINDS NIH HHS / United States
R01 NS036654 / NS / NINDS NIH HHS / United States
5U54-HG003918 / HG / NHGRI NIH HHS / United States