Miranda A, Lahrichi S L, Gulyas J, Koerber S C, Craig A G, Corrigan A, Rivier C, Vale W, Rivier J
Clayton Foundation Laboratories for Peptide Biology, Salk Institute, La Jolla, California 92037, USA.
J Med Chem. 1997 Oct 24;40(22):3651-8. doi: 10.1021/jm970311t.
Hypothesis driven and systematic structure-activity relationship (SAR) investigations have resulted in the development of effective central nervous system (CNS) antagonists of corticotropin (ACTH)-releasing factor (CRF) such as alpha-helical CRF(9-41) and analogues of our assay standard [DPhe12,Nle21,38]hCRF(12-41). On the other hand, equally potent CRF antagonists that block the hypothalamic/pituitary/adrenal (HPA) axis had not been described until recently. Predictive methods, physicochemical measurements (nuclear magnetic resonance spectrometry and circular dichroism spectroscopy), and SAR studies suggest that CRF and its family members (urotensins and sauvagine) assume an alpha-helical conformation when interacting with CRF receptors. To further test this hypothesis, we have systematically scanned the hCRF(9-41) or hCRF(12-41) sequences with an i-(i + 3) bridge consisting of the Glu-Xaa-Xbb-Lys scaffold which we and others had shown could maintain or enhance alpha-helical structure. From this series we have identified seven analogues that are either equipotent to, or 3 times more potent than, the assay standard; in addition, as presented earlier cyclo(30-33)[DPhe12,-Nle21,38,Glu30, Lys33]hCRF(12-41) (astressin) is 32 times more potent than the assay standard in blocking ACTH secretion in vitro (rat pituitary cell culture assay). In vivo, astressin is also significantly more potent than earlier antagonists at reducing hypophysial ACTH secretion in intact stressed or adrenalectomized rats. Since the corresponding linear analogues that were tested are significantly less potent, our interpretation of the increased potency of the cyclic analogues is that the introduction of the side chain to side chain bridging element (Glu30-Lys33, and to a lesser extent that of Glu14-Lys17, Glu20-Lys23, Glu23-Lys26, Glu26-Lys29, Glu28-Lys31, Glu29-Lys32, and Glu33-Lys36) induces and stabilizes in the receptor environment a putative alpha-helical bioactive conformation of the fragment that is not otherwise heavily represented. The effect of the introduction of two favored substitutions [(cyclo(20-23) and cyclo(30-33)] yielded 37 with a potency 8 times that of the assay standard but actually 12 times less than expected if the effect of the two cycles had been multiplicative. These results suggest that the pituitary CRF receptor can discriminate between slightly different identifiable conformations, dramatically illustrating the role that secondary and tertiary structures play in modulating biological signaling through specific protein-ligand interactions.
基于假设驱动和系统的构效关系（SAR）研究，已开发出有效的促肾上腺皮质激素（ACTH）释放因子（CRF）的中枢神经系统（CNS）拮抗剂，如α-螺旋CRF(9 - 41)以及我们的测定标准物[DPhe12,Nle21,38]hCRF(12 - 41)的类似物。另一方面，直到最近才报道了能阻断下丘脑/垂体/肾上腺（HPA）轴的同等效力的CRF拮抗剂。预测方法、物理化学测量（核磁共振光谱法和圆二色光谱法）以及SAR研究表明，CRF及其家族成员（尿紧张素和蛙皮素）在与CRF受体相互作用时呈现α-螺旋构象。为了进一步验证这一假设，我们用由Glu-Xaa-Xbb-Lys支架组成的i-(i + 3)桥对hCRF(9 - 41)或hCRF(12 - 41)序列进行了系统扫描，我们和其他人已表明该支架可以维持或增强α-螺旋结构。从这个系列中，我们鉴定出了7种类似物，它们与测定标准物效力相当或比其强3倍；此外，如前所述，环(30 - 33)[DPhe12,-Nle21,38,Glu30,Lys33]hCRF(12 - 41)（阿斯特辛）在体外阻断促肾上腺皮质激素（ACTH）分泌方面（大鼠垂体细胞培养测定）比测定标准物强32倍。在体内，在完整应激或肾上腺切除的大鼠中，阿斯特辛在减少垂体促肾上腺皮质激素（ACTH）分泌方面也比早期拮抗剂显著更有效。由于所测试的相应线性类似物效力明显较低，我们对环状类似物效力增加的解释是，引入侧链到侧链桥接元件（Glu30-Lys33，以及在较小程度上的Glu14-Lys17、Glu20-Lys23、Glu23-Lys26、Glu26-Lys29、Glu28-Lys31、Glu29-Lys32和Glu33-Lys36）在受体环境中诱导并稳定了该片段假定的α-螺旋生物活性构象，如果没有这些桥接元件，该构象在片段中所占比例不大。引入两个有利取代基[(环(20 - 23)和环(30 - 33)]的效果产生了效力为测定标准物8倍的化合物37，但实际上比如果两个环的效果是相乘时预期的少12倍。这些结果表明，垂体CRF受体可以区分略有不同的可识别构象，显著说明了二级和三级结构在通过特定蛋白质-配体相互作用调节生物信号传导中所起的作用。
