Miranda A, Koerber S C, Gulyas J, Lahrichi S L, Craig A G, Corrigan A, Hagler A, Rivier C, Vale W, Rivier J
Clayton Foundation Laboratories for Peptide Biology, Salk Institute for Biological Studies, La Jolla, California 92037.
J Med Chem. 1994 May 13;37(10):1450-9. doi: 10.1021/jm00036a010.
Corticotropin releasing factor (CRF) is a 41-peptide amide which stimulates the release of ACTH (Vale et al. Science 1981, 213, 1394). CRF has been postulated to assume an alpha-helical conformation upon binding to its pituitary receptor (Hernandez et al. J. Med. Chem. 1993, 36, 2860). We have exploited this hypothesis in the design of a limited series of cyclic analogues and have taken into consideration the effects of side-chain deletion (Alanine scan, Kornreich et al. J. Med. Chem. 1992, 35, 1870) as well as of changes in chirality (Rivier et al. J. Med. Chem. 1993, 36, 2851), with the rationale that side chains necessary for binding could also be replaced by side-chain bridges. In particular, we have used computer modeling to predict likely side chain bridging opportunities and evaluated the effects of such replacements by correlating biological results with those derived from CD spectroscopy. We have synthesized 38 monocyclic peptide amides, competitive antagonists of human/rat CRF, using solid-phase methodology on MBHA resin. After purification by preparative RP-HPLC, the peptides were analyzed by RP-HPLC and capillary zone electrophoresis and characterized by mass spectroscopy and amino acid analysis. CRF antagonists were tested for their ability to interfere with CRF-induced release of ACTH by rat anterior pituitary cells. In most cases, one of the bridge heads was located at a position where substitution by a D-residue was tolerated (i.e., positions 12 and 20). It has become clear that careful optimization of bridge length and chirality is critical. This is best exemplified by the fact that out of the 38 analogues that were synthesized and tested, only two, [cyclo(20-23)[DPhe12,Glu20,Lys23, Nle21,38]h/rCRF12-41 and cyclo(20-23)[DPhe12,Glu20,Orn23,Nle21,38] h/rCRF12-41], were found to be more potent (3 and 2 times, respectively) than [DPhe12,Nle21,38]h/rCRF12-41, the parent compound. Six analogues belonging to two different families were found to be half as potent as the standard, 18 had 2-20% of the potency of the standard, and the others were significantly less potent. CD results of all analogues in 50% TFE (a concentration of TFE that induced nearly maximum helicity of [DPhe12,Nle21,38]h/rCRF12-41) suggest that while helicity may be an important factor for CRF analogue recognition, little correlation is found between percent helicity as determined by spectral deconvolution and biological activity in vitro.
促肾上腺皮质激素释放因子（CRF）是一种41肽酰胺，可刺激促肾上腺皮质激素（ACTH）的释放（Vale等人，《科学》，1981年，213卷，1394页）。据推测，CRF与垂体受体结合后会呈现α-螺旋构象（Hernandez等人，《药物化学杂志》，1993年，36卷，2860页）。我们在设计一系列有限的环状类似物时利用了这一假设，并考虑了侧链缺失（丙氨酸扫描，Kornreich等人，《药物化学杂志》，1992年，35卷，1870页）以及手性变化（Rivier等人，《药物化学杂志》，1993年，36卷，2851页）的影响，其基本原理是结合所需的侧链也可以被侧链桥取代。特别是，我们使用计算机建模来预测可能的侧链桥接机会，并通过将生物学结果与圆二色光谱（CD）光谱得出的结果相关联来评估此类取代的效果。我们使用MBHA树脂上的固相方法合成了38种单环肽酰胺，它们是人类/大鼠CRF的竞争性拮抗剂。通过制备型反相高效液相色谱（RP-HPLC）纯化后，对这些肽进行了反相高效液相色谱和毛细管区带电泳分析，并通过质谱和氨基酸分析进行了表征。测试了CRF拮抗剂干扰大鼠垂体前叶细胞中CRF诱导的促肾上腺皮质激素释放的能力。在大多数情况下，其中一个桥头位于可以耐受D-残基取代的位置（即第12和20位）。很明显，仔细优化桥的长度和手性至关重要。这一点最能通过以下事实体现：在合成和测试的38种类似物中，只有两种，即[环（20-23）[D-苯丙氨酸12，谷氨酸20，赖氨酸23，正亮氨酸21，38]人/大鼠CRF12-41和环（20-23）[D-苯丙氨酸12，谷氨酸20，鸟氨酸23,正亮氨酸21，38]人/大鼠CRF12-41]，被发现比母体化合物[D-苯丙氨酸]12，正亮氨酸21，38]人/大鼠CRF12-41更有效（分别为3倍和2倍）。发现属于两个不同家族的六种类似物的效力是标准物的一半，18种具有标准物效力的2%-20%，其他的效力则明显更低。所有类似物在5-%三氟乙醇（TFE）（一种诱导[D-苯丙氨酸12，正亮氨酸21，38]人/大鼠CRF12-41几乎最大螺旋度的TFE浓度）中的CD结果表明，虽然螺旋度可能是CRF类似物识别的一个重要因素，但通过光谱解卷积确定的螺旋度百分比与体外生物学活性之间几乎没有相关性。
