Kapurniotu A, Taylor J W
Rutgers University, Department of Chemistry, Piscataway, New Jersey 08855-0939.
J Med Chem. 1995 Mar 3;38(5):836-47. doi: 10.1021/jm00005a011.
The conformational and pharmacological effects of the introduction of conformational constraints in the form of i-(i + 4) lactam-bridges in the potential amphiphilic alpha-helical region (8-21) of human calcitonin (hCT) were studied. The following three cyclic hCT analogues were synthesized: cyclo17,21-[Lys17,Asp21]hCT (1), cyclo17,21- [Asp17,Lys21]hCT (2) and cyclo10,14-[Lys10,-Asp14]hCT (3). For their syntheses, solid-phase methodology was used in combination with either direct side chain to side chain cyclization on the solid support or a segment-condensation strategy. Circular dichroism studies in aqueous buffer, pH 7.0, indicated that the conformational effects were different for each lactam bridge introduced. Significant induction of alpha-helical structure was observed only for peptide 3. In contrast, peptide 1 and hCT had similar CD spectra, indicative of mixed disordered and beta-sheet conformations, and peptide 2 had a weaker spectrum consistent with the formation of a more ordered but nonhelical structure. In rat brain receptor binding assays, peptide 2 showed a nearly 80-fold higher potency than hCT or peptides 1 and 3. All three analogues stimulated adenylyl cyclase in the rat kidney membrane at 5-fold lower concentrations than hCT and with similar maximal effects. In vivo hypocalcemic assays, performed in mice by analysis of serum calcium levels 1 h after sc injection, indicated that peptide 2 had similar maximal effects to hCT and was 10-20 times more potent than hCT at doses giving half-maximal effects. In contrast, peptides 1 and 3 were not significantly more potent than hCT. Our findings indicate compatibility of all three lactam bridges and, most probably, also the amphiphilic alpha-helix, with the pharmacological activities of hCT. However, the properties of peptide 2 also suggest that another conformation, possibly a type I beta-turn involving residues 17-20, may play an important role. A multistep mechanism of receptor recognition by hCT that might account for these results is discussed.
研究了在人降钙素（hCT）潜在的两亲性α-螺旋区域（8 - 21）引入i-(i + 4)内酰胺桥形式的构象限制所产生的构象和药理学效应。合成了以下三种环化hCT类似物：环[17,21]-[赖氨酸17，天冬氨酸21]hCT（1）、环[17,21]-[天冬氨酸17，赖氨酸21]hCT（2）和环[10,14]-[赖氨酸10，天冬氨酸14]hCT（3）。对于它们的合成，采用了固相方法，结合在固相载体上直接进行侧链到侧链环化或片段缩合策略。在pH 7.0的水性缓冲液中进行的圆二色性研究表明，引入的每个内酰胺桥的构象效应不同。仅在肽3中观察到显著的α-螺旋结构诱导。相比之下，肽1和hCT具有相似的CD光谱，表明存在混合的无序和β-折叠构象，而肽2的光谱较弱，这与形成更有序但非螺旋结构一致。在大鼠脑受体结合试验中，肽2的效力比hCT或肽1和3高近80倍。所有三种类似物在大鼠肾膜中刺激腺苷酸环化酶的浓度比hCT低5倍，且具有相似的最大效应。在小鼠体内进行的低血钙试验中，通过皮下注射1小时后分析血清钙水平，结果表明肽2与hCT具有相似的最大效应，并且在产生半数最大效应的剂量下比hCT强10 - 20倍。相比之下，肽1和3的效力并不比hCT显著更强。我们的研究结果表明，所有三种内酰胺桥以及很可能两亲性α-螺旋与hCT的药理活性具有兼容性。然而，肽2的性质也表明，另一种构象，可能是涉及残基17 - 20的I型β-转角，可能起重要作用。讨论了hCT受体识别的多步机制，该机制可能解释这些结果。
