Kirby D A, Boublik J H, Rivier J E
Clayton Foundation Laboratories for Peptide Biology, Salk Institute, La Jolla, California 92037.
J Med Chem. 1993 Nov 26;36(24):3802-8. doi: 10.1021/jm00076a007.
In an effort to gain insight into the bioactive conformation of neuropeptide Y upon interaction with its receptors, all single-point D-amino acid substituted NPY analogues were prepared, and their Y1 and Y2 receptor binding affinities were evaluated using the human neuroblastoma cell lines, SK-N-MC and SK-N-BE2, respectively. Solid-phase synthesis (Boc strategy) followed by preparative HPLC purification produced analogues of high purity that were characterized by RP-HPLC, AAA, LSIMS, CZE, and optical rotation. Of the 37 isomers (a naturally occurring glycine at position 9 was replaced by Ala and D-Ala), Y1 receptor binding was most perturbed by chiral inversion of residues at the C-terminus (residues 20, 27, 29-35, Ki > or = 300 nM). Substitutions at residues 2-5, 28, and 36 had Ki values ranging from 40 to 260 nM. Substitutions at all other positions yielded analogues with affinities ranging from 1.5 to 20 nM. Binding affinities to the Y2 class of receptors all measured in the low or sub-nanomolar concentrations, with the exception of C-terminally modified isomers (residues 30-35). Only [D-Arg33]- and [D-Gln34]NPY displayed no measurable binding affinity to Y2 receptors at the highest concentration tested (1000 nM). Representative analogues were selected on the basis of their binding affinities and position in the sequence for structural analysis using circular dichroism (CD) spectroscopy. Of the nine peptide evaluated ([D-Pro5]-, [Ala9]-, [D-Glu10]-, [D-Asp11]-, [D-Ala18]-, [D-Tyr20]-, [D-Tyr27]-, and [D-Arg33]NPY), only [D-Tyr27]NPY expressed a definitive correlation between loss of binding affinity and disruption of secondary structure by having the propensity to form beta-sheets at the expense of alpha-helical content. It was concluded that although the incorporation of a single D-amino acid within the sequence of NPY may confer a conformational perturbation, the receptor interaction was only affected when certain critical residues were modified, findings that provide a basis for the identification of the binding pharmacophore of NPY.
为深入了解神经肽Y与受体相互作用时的生物活性构象，制备了所有单点D - 氨基酸取代的NPY类似物，并分别使用人神经母细胞瘤细胞系SK - N - MC和SK - N - BE2评估它们与Y1和Y2受体的结合亲和力。通过固相合成（Boc策略）并经制备型HPLC纯化得到高纯度类似物，这些类似物通过反相高效液相色谱（RP - HPLC）、氨基酸分析（AAA）、液相二次离子质谱（LSIMS）、毛细管区带电泳（CZE）和旋光性进行表征。在37种异构体中（第9位天然存在的甘氨酸被丙氨酸和D - 丙氨酸取代），C末端残基（残基20、27、29 - 35）的手性反转对Y1受体结合的影响最大（Ki≥300 nM）。残基2 - 5、28和36处的取代，其Ki值范围为40至260 nM。所有其他位置的取代产生的类似物亲和力范围为1.5至20 nM。除了C末端修饰的异构体（残基30 - 35）外，与Y2类受体的结合亲和力均在低或亚纳摩尔浓度范围内测定。只有[D - Arg33] - 和[D - Gln34]NPY在测试的最高浓度（1000 nM）下对Y2受体没有可测量的结合亲和力。根据结合亲和力和序列位置选择代表性类似物，用于使用圆二色性（CD）光谱进行结构分析。在评估的9种肽（[D - Pro5] - 、[Ala9] - 、[D - Glu10] - 、[D - Asp11] - 、[D - Ala18] - 、[D - Tyr20] - 、[D - Tyr27] - 和[D - Arg33]NPY）中，只有[D - Tyr27]NPY在结合亲和力丧失与二级结构破坏之间表现出明确的相关性，它倾向于形成β - 折叠而牺牲α - 螺旋含量。得出的结论是，虽然在NPY序列中掺入单个D - 氨基酸可能会引起构象扰动，但只有当某些关键残基被修饰时，受体相互作用才会受到影响，这些发现为确定NPY的结合药效基团提供了基础。
