Characterization of the Binding Site for a Novel Class of Noncompetitive α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Receptor Antagonists

  1. Frank S. Menniti,
  2. Bertrand L. Chenard,
  3. Mary B. Collins,
  4. Mary F. Ducat,
  5. Mark L. Elliott,
  6. Frank E. Ewing,
  7. Jianhua I. Huang,
  8. Kristin A. Kelly,
  9. John T. Lazzaro,
  10. Martin J. Pagnozzi,
  11. James L. Weeks,
  12. Willard M. Welch and
  13. W. Frost White
  1. Pfizer Global Research and Development, Groton, Connecticut

    Abstract

    The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor is an ionotropic glutamate receptor that mediates fast excitatory synaptic transmission throughout the central nervous system. In addition to the glutamate binding site, allosteric modulatory sites on the receptor are inferred from the ability of synthetic compounds to affect channel function without interaction with the glutamate binding site. We have identified a novel class of potent, noncompetitive AMPA receptor antagonists typified by CP-465,022 and CP-526,427. The latter compound was radiolabeled and used to elucidate the pharmacology of one allosteric modulatory site. [3H]CP-526,427 labels a single binding site in rat forebrain membranes with aK d value of 3.3 nM and aB max of 7.0 pmol/mg of protein. The [3H]CP-526,427 binding site does not seem to interact directly with the glutamate binding site but overlaps with that for another class of AMPA receptor antagonists, the 2,3-benzodiazepines. This binding site is distinct from that for the antagonist Evans blue and for several classes of compounds that modulate AMPA receptor desensitization. These results indicate the existence of at least two physically distinct allosteric sites on the AMPA receptor through which channel activity or desensitization is modulated.

    Footnotes

    • Send reprint requests to: Dr. Frank S. Menniti, Pfizer Inc., Eastern Point Road, Groton, CT 06340. E-mail:mennitifs{at}groton.pfizer.com

    • This work was supported by Pfizer, Inc.

    • Abbreviations:
      AMPA
      α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
      NMDA
      N-methyl-d-aspartate
      NBQX
      2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline
      GYKI-52,466
      4-(8-methyl-9H-1,3-dioxa-6,7-diaza-cyclohepta[f]inden-5-yl)-phenylamine
      BCP-1
      1-(1,3-benzodioxol-5-ylcarbonyl)piperidine
      CX-516
      1-(6-quinoxalinylcarbonyl)-piperidine
      Co 102,659
      7-(1-ethyl-propyl)-5-phenyl-7,9-dihydro-1,3-dioxa-6,7-diaza-cyclohepta[f]inden-8-one
      Co 102,685
      7-cyclohexyl-5-phenyl-7,9-dihydro-1,3-dioxa-6,7-diaza-cyclohepta[f]inden-8-one
      CP-465,022
      3-(2-Chloro-phenyl)-2-[2-(6-diethylaminomethyl-pyridin-2-yl)-vinyl]-6-fluoro-3H-quinazolin-4-one
      CP-526,427
      2-{2-[3-(2-Chloro-phenyl)-6-fluoro-4-oxo-3,4-dihydro-quinazolin-2-yl]-vinyl}-nicotinonitrile
      Co 102,581
      5-phenyl-7,9-dihydro-1,3-dioxa-6,7-diaza-cyclohepta[f]inden-8-one
      YM-90K
      6-(1H-imidazol-1-yl)-7-nitro-2,3(1h,4h)-quinoxalinedione hydrochloride
      BSS
      balanced salt solution
      RFUs
      relative fluorescent units
      CP-471,236
      6-Fluoro-3-(2-methyl-pyridin-3-yl)-2-[2-(2-methyl-thiazol-4-yl)-vinyl]-3H-quinazolin-4-one
      MK-801
      (+)-5-methyl-10,11-dihydro-5-H-dibenzo[a,d]cycloheptan-5,10-imine
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    1. Molecular Pharmacology December 1, 2000 vol. 58 no. 6 1310-1317
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