title: Characterization of the binding and activity of a high affinity, pseudoirreversible morpholino tachykinin NK1 receptor antagonist.
abstract: 2(S)-((3,5-Bis(trifluoromethyl)benzyl)-oxy)-3(S)-phenyl-4-((3-oxo-1,2,4- triazol-5-yl)methyl)morpholine (L-742,694) is a selective morpholino tachykinin NK1 receptor antagonist that inhibits the binding of 125I-substance P to the human tachykinin NK1 receptor with a Kd = 37 pM. Increasing concentrations of L-742,694 added to cells 15 min prior to agonist progressively increase the apparent EC50 of substance P for inducing the synthesis of inositol phosphate in Chinese hamster ovary (CHO) cells expressing human tachykinin NK1 receptor and decrease the maximal level of stimulation observed. In contrast, addition of substance P and L-742,694 to the cells at the same time results in an increase in the EC50 for substance P with no decrease in the maximal level of stimulation. The compound also decreases the apparent number of binding sites for 125I-substance P observed by Scatchard analysis. Analysis of the binding of [3H]L-742,694 to the tachykinin NK1 receptor shows that it associates with the receptor with k(a) = 3.98 x 10(8) M(-1) min(-1), and dissociates with k(d) = 0.026 min(-1) and t1/2 = 27 min at 22 degrees C. The slow rate of dissociation of L-742,694 from the tachykinin NK1 receptor and the observation that altering the order of addition of antagonist and substance P attenuates the effect of the antagonist on the maximal activation suggest that L-742,694 is a competitive antagonist that can behave as a pseudoirreversible antagonist under some experimental conditions. L-742,694 has reduced affinity for tachykinin NK1 receptors in which alanine has been substituted for Gln165, His197 or His265 in transmembrane helices 4, 5 and 6, respectively. These three residues have previously been shown to be present in the binding site of tachykinin NK1 receptor antagonists of several structural classes. In addition, L-742,694 inhibits binding of the quinuclidine antagonist (2S,3S)-cis-2-(diphenyl methyl)-N-[(2-iodophenyl)-methyl]-1-azabicyclo[2.2.2]octane 3-amine ([125I]L-703,606) with the same affinity as it inhibits binding of 125I-substance P. These data indicate that L-742,694 binds to the same site within the transmembrane domain of the receptor as previously described competitive antagonists.
authors.affiliation: Department of Molecular Pharmacology, Merck Research Laboratories, Rahway, NJ 07065, USA. peggy_cascieri@merck.com
authors.forename: M A
authors.initials: MA
authors.lastname: Cascieri
authors.forename: E
authors.initials: E
authors.lastname: Ber
authors.forename: T M
authors.initials: TM
authors.lastname: Fong
authors.forename: J J
authors.initials: JJ
authors.lastname: Hale
authors.forename: F
authors.initials: F
authors.lastname: Tang
authors.forename: L L
authors.initials: LL
authors.lastname: Shiao
authors.forename: S G
authors.initials: SG
authors.lastname: Mills
authors.forename: M
authors.initials: M
authors.lastname: MacCoss
authors.forename: S
authors.initials: S
authors.lastname: Sadowski
authors.forename: M R
authors.initials: MR
authors.lastname: Tota
authors.forename: C D
authors.initials: CD
authors.lastname: Strader
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D006801:Humans; D007700:Kinetics; D009025:Morpholines / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed; D064729:Neurokinin-1 Receptor Antagonists; D018040:Receptors, Neurokinin-1 / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera; D013373:Substance P / Q000378:metabolism / Q000494:pharmacology
chemical_list: C099406:L 742694; D009025:Morpholines; D064729:Neurokinin-1 Receptor Antagonists; D018040:Receptors, Neurokinin-1; D011994:Recombinant Proteins; D013373:Substance P