Cascieri M A, Ber E, Fong T M, Hale J J, Tang F, Shiao L L, Mills S G, MacCoss M, Sadowski S, Tota M R, Strader C D
Department of Molecular Pharmacology, Merck Research Laboratories, Rahway, NJ 07065, USA.
Eur J Pharmacol. 1997 May 1;325(2-3):253-61. doi: 10.1016/s0014-2999(97)00122-2.
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.
2(S)-((3,5-双(三氟甲基)苄基)-氧基)-3(S)-苯基-4-((3-氧代-1,2,4-三唑-5-基)甲基)吗啉(L-742,694)是一种选择性吗啉代速激肽NK1受体拮抗剂，其抑制125I- P物质与人速激肽NK1受体的结合，解离常数Kd = 37 pM。在激动剂作用前15分钟向细胞中加入浓度递增的L-742,694，可使P物质在表达人速激肽NK1受体的中国仓鼠卵巢(CHO)细胞中诱导肌醇磷酸合成的表观半数有效浓度(EC50)逐渐升高，并降低观察到的最大刺激水平。相反，同时向细胞中加入P物质和L-742,694会导致P物质的EC50升高，而最大刺激水平没有降低。该化合物还会减少通过Scatchard分析观察到的125I- P物质的表观结合位点数。对[3H]L-742,694与速激肽NK1受体结合的分析表明，它与受体结合的结合速率常数k(a) = 3.98×10(8) M(-1) min(-1)，在22℃下解离速率常数k(d) = 0.026 min(-1)，半衰期t1/2 = 27分钟。L-742,694从速激肽NK1受体解离的速率较慢，并且改变拮抗剂和P物质添加顺序会减弱拮抗剂对最大激活的作用，这表明L-742,694是一种竞争性拮抗剂，在某些实验条件下可表现为拟不可逆拮抗剂。L-742,694对速激肽NK1受体的亲和力降低，其中在跨膜螺旋4、5和6中，丙氨酸分别取代了Gln165、His197或His265。先前已表明这三个残基存在于几种结构类型的速激肽NK1受体拮抗剂的结合位点中。此外，L-742,694抑制奎宁环拮抗剂(2S,3S)-顺式-2-(二苯基甲基)-N-[(2-碘苯基)-甲基]-1-氮杂双环[2.2.2]辛烷3-胺([125I]L-703,606)结合的亲和力与其抑制125I- P物质结合的亲和力相同。这些数据表明，L-742,694与受体跨膜结构域内的同一位点结合，如同先前描述的竞争性拮抗剂一样。
