Lovejoy D A, Corrigan A Z, Nahorniak C S, Perrin M H, Porter J, Kaiser R, Miller C, Pantoja D, Craig A G, Peter R E
Clayton Foundation Laboratories for Peptide Biology, Salk Institute, La Jolla, CA 92037, USA.
Regul Pept. 1995 Dec 14;60(2-3):99-115. doi: 10.1016/0167-0115(95)00116-6.
Three natural forms of vertebrate gonadotropin-releasing hormone (GnRH) provided the structural basis upon which to design new GnRH agonists: [His5,Trp7,Leu8]-GnRH, dogfish (df) GnRH; [His5,Asn8]-GnRH, catfish (cf) GnRH; and [His5,Trp7,Tyr8]-GnRH, chicken (c) GnRH-II. The synthetic peptides incorporated the position 6 dextro (D)-isomers D-arginine (D-Arg) or D-naphthylalanine (D-Nal) in combination with an ethylamide substitution of position 10. The in vitro potencies for LH and FSH release of these analogues were assessed using static cultures of rat anterior pituitary cells. Efficacious peptides were examined for their gonadotropin-II and growth hormone releasing abilities from perifused goldfish pituitary fragments. Rat LH and FSH release was measured using homologous radioimmunoassays, whereas goldfish growth hormone and gonadotropin-II release were determined using heterologous carp hormone radioimmunoassays. The receptor binding of the most potent analogues was determined in bovine pituitary membrane preparations. Substitution of D-Nal6 into [His5,Asn8]-GnRH increased the potency over 2200-fold compared with the native ligand (cfGnRH) in cultured rat pituitary cells. This was equivalent to a 55-fold greater potency than that of the native mammal (m) GnRH peptide. Substitution of D-Nal6 or D-Arg6 into dfGnRH or cGnRH-II resulted in potencies that were related to the overall hydrophobicity of the analogues. The [D-Nal6,Pro9NEt]-cfGnRH bound to the bovine membrane preparation with an affinity statistically similar to that of [D-Nal6,Pro9NEt]-mGnRH (kd = 0.40 +/- 0.04 and 0.55 +/- 0.10 nM, respectively) in cultured rat pituitary cells. All analogues tested released the same ratio of FSH to LH. In goldfish, the analogues did not possess superagonistic activity but instead desensitized the pituitary fragments at lower analogue doses than that of the sGnRH standard suggesting differences in receptor affinity or signal transduction.
脊椎动物促性腺激素释放激素（GnRH）的三种天然形式为设计新型GnRH激动剂提供了结构基础：[His5,Trp7,Leu8]-GnRH，即鲨鱼（df）GnRH；[His5,Asn8]-GnRH，即鲶鱼（cf）GnRH；以及[His5,Trp7,Tyr8]-GnRH，即鸡（c）GnRH-II。合成肽在第6位引入了右旋（D）-异构体D-精氨酸（D-Arg）或D-萘丙氨酸（D-Nal），并在第10位进行了乙酰胺取代。使用大鼠垂体前叶细胞的静态培养物评估这些类似物对促黄体生成素（LH）和促卵泡生成素（FSH）释放的体外效力。对有效的肽进行检测，以评估其从灌注的金鱼垂体片段释放促性腺激素-II和生长激素的能力。使用同源放射免疫测定法测量大鼠LH和FSH的释放，而使用异源鲤鱼激素放射免疫测定法测定金鱼生长激素和促性腺激素-II的释放。在牛垂体膜制剂中测定最有效类似物的受体结合情况。在培养的大鼠垂体细胞中，将D-Nal6取代[His5,Asn8]-GnRH后，其效力比天然配体（cfGnRH）提高了2200倍以上。这相当于比天然哺乳动物（m）GnRH肽的效力高55倍。将D-Nal6或D-Arg6取代dfGnRH或cGnRH-II后，效力与类似物的整体疏水性有关。在培养的大鼠垂体细胞中，[D-Nal6,Pro9NEt]-cfGnRH与牛膜制剂的结合亲和力在统计学上与[D-Nal6,Pro9NEt]-mGnRH相似（kd分别为0.40±0.04和0.55±0.10 nM）。所有测试的类似物释放的FSH与LH的比例相同。在金鱼中，这些类似物不具有超激动活性，而是在比sGnRH标准更低的类似物剂量下使垂体片段脱敏，这表明受体亲和力或信号转导存在差异。
