Meini Stefania, Catalani Claudio, Bellucci Francesca, Cucchi Paola, Giuliani Sandro, Zappitelli Sabrina, Rotondaro Luigi, Pasqui Franco, Guidi Antonio, Altamura Maria, Giolitti Alessandro, Maggi Carlo Alberto
Department of Pharmacology, Menarini Ricerche S.p.A., via Rismondo 12A, Florence, Italy.
Eur J Pharmacol. 2005 Jun 1;516(2):104-11. doi: 10.1016/j.ejphar.2005.04.033.
The pharmacological outline of a novel and original antagonist at the human tachykinin NK2 receptor is presented, namely MEN13510 (N-N'-bis-[2-(1H-indol-3-yl)-ethyl]-N,N'-bis-(3-thiomorpholin-4-yl-propyl)-phthalamide). MEN13510 retained nanomolar affinity for the human tachykinin NK2 receptor (Ki 6.4 nM), and micromolar affinity for the human tachykinin NK1 and NK3 receptors. A competitive antagonism is indicated by the Schild analysis (pK(B) 7.8, slope -0.94) of concentration-response curves of NKA induced inositolphosphates accumulation in Chinese hamster ovary (CHO) cells expressing the human NK2 receptor in the presence of MEN13510 (30-300 nM concentration range). The MEN13510 interaction with the human NK2 receptor was evaluated by means of heterologous inhibition binding experiments, by using agonist and antagonist radioligands ([125I]NKA, [3H]nepadutant, [3H]saredutant) at a series of mutant receptors having single aminoacidic substitutions of residues located in transmembrane (TM) segments 3, 4, 5, 6, and 7. MEN13510 affinity was not affected by the mutations in TM 3 and 4 (Q109A, F112A, T171A, C167G), and it was reduced by 10-fold at the I202F mutant, but not at the Y206A (TM4). Amongst the investigated mutants bearing the mutated residues in TM6 (F270A, Y266F, W263A) only F270A decreased the MEN13510 affinity by 7-fold. Even mutations in TM7 did reduce MEN13510 affinity by 32-fold (Y289T, but not Y289F) and 13-fold (F293A). Studied mutations represent the human tachykinin NK2 receptor discriminants involved in the binding of previously reported peptidic and nonpeptidic antagonists, against which results obtained with MEN13510 are compared. Results indicate that the binding site of this antagonist is, at least in part, overlapping to that described for NKA or saredutant. Finally we show that MEN13510 retains nanomolar affinity for the recently discovered splice variant of the human tachykinin NK2 receptor, namely beta isoform, as it has been described for the nonpeptide antagonist saredutant.
本文介绍了一种新型、原创的人类速激肽NK2受体拮抗剂的药理学概况，即MEN13510（N-N'-双-[2-(1H-吲哚-3-基)-乙基]-N,N'-双-(3-硫代吗啉-4-基丙基)-邻苯二甲酰胺）。MEN13510对人类速激肽NK2受体保持纳摩尔亲和力（Ki 6.4 nM），对人类速激肽NK1和NK3受体保持微摩尔亲和力。在表达人类NK2受体的中国仓鼠卵巢（CHO）细胞中，MEN13510（浓度范围为30 - 300 nM）存在时，NKA诱导的肌醇磷酸积累浓度-反应曲线的Schild分析（pK(B) 7.8，斜率 -0.94）表明存在竞争性拮抗作用。通过异源抑制结合实验，使用激动剂和拮抗剂放射性配体（[125I]NKA、[3H]奈帕他定、[3H]沙瑞他定）在一系列具有位于跨膜（TM）片段3、4、5、6和7的单个氨基酸取代的突变受体上评估MEN13510与人类NK2受体的相互作用。MEN13510的亲和力不受TM 3和4中的突变（Q109A、F112A、T171A、C167G）影响，在I202F突变体处降低了10倍，但在Y206A（TM4）处未降低。在TM6中带有突变残基的研究突变体（F270A、Y266F、W263A）中，只有F270A使MEN13510的亲和力降低了7倍。即使TM7中的突变也使MEN13510的亲和力分别降低了32倍（Y289T，但不是Y289F）和13倍（F293A）。所研究的突变代表了参与先前报道的肽类和非肽类拮抗剂结合的人类速激肽NK2受体判别因素，并将其与MEN13510获得的结果进行了比较。结果表明，该拮抗剂的结合位点至少部分与NKA或沙瑞他定所描述的位点重叠。最后我们表明，MEN13510对最近发现的人类速激肽NK2受体剪接变体，即β异构体，保持纳摩尔亲和力，正如对非肽拮抗剂沙瑞他定所描述的那样。
