Audinot V, Lahaye C, Suply T, Beauverger P, Rodriguez M, Galizzi J P, Fauchère J L, Boutin J A
Division de Pharmacologie Moléculaire et Cellulaire, Institut de Recherches Servier, 125 chemin de Ronde, 78290 Croissy sur Seine, France.
Br J Pharmacol. 2001 Jun;133(3):371-8. doi: 10.1038/sj.bjp.0704085.
Shortened, more stable and weakly hydrophobic analogues of melanin-concentrating hormone (MCH) were searched as candidates for radioiodination. Starting from the dodecapeptide MCH(6 - 17), we found that: (1) substitution of Tyr(13) by a Phe residue; (2) addition of a 3-iodo-Tyr residue at the N-terminus; and (3) addition of a hydrophilic spacer 8-amino-3,6-dioxyoctanoyl between the 3-iodo-Tyr and MCH(6 - 17) (compound S36057), led to an agonist more potent than MCH itself in stimulating [35S]-GTPgammaS binding at membranes from HEK293 cells stably expressing the human MCH receptor. Specific binding of [125I]-S36057 was found in HEK293 and CHO cell lines stably expressing the human MCH receptor. This radioligand recognized a similar number of binding sites (ca. 800 fmol mg(-1)) than [125I]-[3-iodo Tyr(13)]-MCH. However, the K(D) for [125I]-S36057 obtained from saturation studies (0.037 nM) or from binding kinetics (0.046 nM) was at least 10 fold higher to that of [125I]-[3-iodo Tyr(13)]-MCH (0.46 nM). Affinities determined for a series of MCH analogues were similar with both radioligands, S36057 being the most potent compound tested (K(i)=0.053 nM). Finally, [125I]-S36057 also potently labelled the MCH receptor in membranes from whole rat brain (K(D) 0.044 nM, B(max)=11 fmol mg(-1)). In conclusion, [125I]-S36057 is a more potent and more stable radioligand than [125I]-[3-iodo Tyr(13)]-MCH that will represent a reliable tool for binding assays in the search of novel MCH ligands. It should also provide great help for autoradiographic studies of the MCH receptor distribution in the central nervous system.
人们寻找了黑色素聚集激素（MCH）的缩短、更稳定且疏水性较弱的类似物作为放射性碘化的候选物。从十二肽MCH(6 - 17)开始，我们发现：（1）用苯丙氨酸残基取代Tyr(13)；（2）在N端添加一个3 - 碘酪氨酸残基；（3）在3 - 碘酪氨酸和MCH(6 - 17)之间添加一个亲水性间隔基8 - 氨基 - 3,6 - 二氧辛酰基（化合物S36057），得到了一种激动剂，在刺激稳定表达人MCH受体的HEK293细胞膜上的[35S]-GTPγS结合方面比MCH本身更有效。在稳定表达人MCH受体的HEK293和CHO细胞系中发现了[125I]-S36057的特异性结合。这种放射性配体识别的结合位点数与[125I]-[3 - 碘酪氨酸(13)]-MCH相似（约800 fmol mg(-1)）。然而，通过饱和研究（0.037 nM）或结合动力学（0.046 nM）获得的[125I]-S36057的K(D)比[125I]-[3 - 碘酪氨酸(13)]-MCH的K(D)（0.46 nM）至少高10倍。对一系列MCH类似物测定的亲和力与两种放射性配体相似，S36057是测试的最有效化合物（K(i)=0.053 nM）。最后，[125I]-S36057也能有效地标记全大鼠脑细胞膜中的MCH受体（K(D) 0.044 nM，B(max)=11 fmol mg(-1)）。总之，[125I]-S36057是一种比[125I]-[3 - 碘酪氨酸(13)]-MCH更有效、更稳定的放射性配体，将成为寻找新型MCH配体的结合测定的可靠工具。它也应该为中枢神经系统中MCH受体分布的放射自显影研究提供很大帮助。
