Kirby D A, Koerber S C, Craig A G, Feinstein R D, Delmas L, Brown M R, Rivier J E
Clayton Foundation Laboratories for Peptide Biology, Salk Institute, La Jolla, California 92037.
J Med Chem. 1993 Feb 5;36(3):385-93. doi: 10.1021/jm00055a010.
To further elucidate the minimum bioactive conformation of neuropeptide Y (NPY), a series of truncated and conformationally constrained analogues has been prepared. The synthesis and purification of these peptides was achieved using routine laboratory strategies and techniques. Parent molecules consisted of the native NPY N-terminal 1-4 and C-terminal 25-36 segments, having the residue 5-24 core replaced by either a single flexible omega-aminoalkanoic acid, or a more rigid Pro-Gly or Pro-DAla sequence which was expected to constrain a putative turn, and allow the N- and C-termini to align. Cross-linking between residues 2 and 27 through lactamization using side-chain length and chirality suggested by computer simulations, resulted in cyclo-(2/27)-des-AA7-24[Glu2,Gly6,DDpr27]NPY that exhibited very high affinity (Ki = 0.3 versus 0.3 nM for NPY) for the Y2 receptor using SK-N-BE2 human neuroblastoma cells, yet very low affinity for the Y1 receptor using SK-N-MC human neuroblastoma cells (Ki = 130 versus 2.0 nM for NPY). The added constraint resulting from bridging in this analogue as well as in others suggested that the combination of the deletion of residues 5-24 and the introduction of an internal ring produced exclusive selectivity for the Y2 receptor with little or no loss of affinity. The tolerance of structural recognition was further demonstrated as a second ring was introduced which was expected to constrain the amphiphilic alpha-helix, resulting in the full Y2 agonist dicyclo (2/27,28/32)-des-AA7-24 [Glu2,32,DAla6,DDpr27,Lys28]NPY. Improvement of Y1 binding activity was achieved only by including more residues (des-AA10-17) in the central PP-fold region, while allowing limited flexibility of the termini. Although the length of the bridge seemed to have little effect on binding potency, changes in the location of and chirality at the bridgehead resulted in analogues with different binding affinities. Combination of optimum structural modifications resulted in cyclo-(7/21)-des-AA10-18[Cys7,21]NPY, an analogue shortened by 25% but retaining comparable binding properties to that of native NPY at Y1 and Y2 receptor types (Ki = 5.1 and 1.3 nM, respectively).
为了进一步阐明神经肽Y（NPY）的最小生物活性构象，已制备了一系列截短的和构象受限的类似物。这些肽的合成和纯化是使用常规实验室策略和技术完成的。母体分子由天然NPY的N端1 - 4片段和C端25 - 36片段组成，其5 - 24位残基核心被单个柔性ω-氨基链烷酸或更刚性的Pro - Gly或Pro - DAla序列取代，预计这些序列会限制一个假定的转角，并使N端和C端对齐。通过计算机模拟建议的侧链长度和手性进行内酰胺化，使2位和27位残基之间交联，得到环（2/27）-去AA7 - 24[Glu2，Gly6，DDpr27]NPY，使用SK - N - BE2人神经母细胞瘤细胞时，它对Y2受体表现出非常高的亲和力（Ki = 0.3，而NPY为0.3 nM），但使用SK - N - MC人神经母细胞瘤细胞时对Y1受体的亲和力非常低（Ki = 130，而NPY为2.0 nM）。该类似物以及其他类似物中因桥连产生的额外限制表明，删除5 - 24位残基并引入内环的组合产生了对Y2受体的专属选择性，且亲和力几乎没有损失。当引入第二个环以限制两亲性α-螺旋时，进一步证明了结构识别的耐受性，从而得到了完全的Y2激动剂二环（2/27，28/32）-去AA7 - 24 [Glu2,32，DAla6，DDpr27，Lys28]NPY。只有通过在中央PP折叠区域包含更多残基（去AA10 - 17），同时允许末端有有限的柔性，才能实现Y1结合活性的改善。虽然桥的长度似乎对结合效力影响不大，但桥头位置和手性的变化导致了具有不同结合亲和力的类似物。最佳结构修饰的组合产生了环（7/21）-去AA10 - 18[Cys7,21]NPY，该类似物缩短了25%，但在Y1和Y2受体类型上保留了与天然NPY相当的结合特性（Ki分别为5.1和1.3 nM）。
