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

Research output: Contribution to journalJournal articleResearchpeer-review

  • Kasper Bø Hansen
  • Praseeda Mullasseril
  • Sara Dawit
  • Natalie L Kurtkaya
  • Hongjie Yuan
  • Katie M Vance
  • Anna G Orr
  • Trine Kvist
  • Kevin K Ogden
  • Phuong Le
  • Kimberly M Vellano
  • Iestyn Lewis
  • Serdar Kurtkaya
  • Yuhong Du
  • Min Qui
  • T J Murphy
  • James P Snyder
  • Bräuner-Osborne, Hans
  • Stephen F Traynelis
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.
Original languageEnglish
JournalJournal of Pharmacology and Experimental Therapeutics
Issue number3
Pages (from-to)650-662
Publication statusPublished - 2010

Bibliographical note

Keywords: Aniline 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

ID: 21771997