Schiller P W, Berezowska I, Nguyen T M, Schmidt R, Lemieux C, Chung N N, Falcone-Hindley M L, Yao W, Liu J, Iwama S, Smith A B, Hirschmann R
Laboratory of Chemical Biology and Peptide Research, Clinical Research Institute of Montréal, 110 Pine Avenue West, Montréal, Quebec, Canada.
J Med Chem. 2000 Feb 24;43(4):551-9. doi: 10.1021/jm990461z.
Recently we reported using minilibraries to replace Lys(9) [somatostatin (SRIF) numbering] of the potent somatostatin agonist L-363,301 (c[-Pro-Phe-D-Trp-Lys-Thr-Phe-]) to generate the potent neurokinin receptor (NK-1) antagonist c[-Pro-Phe-D-Trp-p-F-Phe-Thr-Phe-]. This novel cyclic hexapeptide did not bind the SRIF receptor. Thus, a single mutation converted L-363,301, a SRIF agonist with potency ca. 2-8 times the potency of SRIF in laboratory animals,(24) into a selective NK-1 receptor antagonist with an IC(50) of 2 nM in vitro. During the screening of the same libraries for ligands of the delta-opioid receptor, we identified four compounds (1-4) which represent a new class of delta-opioid antagonists, some of which were also NK-1 receptor antagonists. The most potent delta-opioid antagonist, c[-Pro-1-Nal-D-Trp-Tyr-Thr-Phe-] (2), showed a K(e) value of 128 nM in the mouse vas deferens assay and a delta-receptor binding affinity constant of 152 nM in the rat brain membrane binding assay. These results are of interest because they represent a novel class of delta-opioid antagonists and, like two previously reported delta-opioid antagonists, they lack a positive charge. To examine further the requirement for a positive charge in the delta-opioid ligands, we prepared two analogues of the beta-casomorphin-derived mixed mu-agonist/delta-antagonist, H-Dmt-c[-D-Orn-2-Nal-D-Pro-Gly-] (7), in which we eliminated the positive charge either through formylation of the primary amino group (5) or by the deletion of this N-terminal amino group (6). These latter compounds proved to be delta-opioid antagonists with K(e) values in the 16-120 nM range, as well as fairly potent mu-opioid antagonists (K(e) approximately 200 nM). These six compounds provide the most convincing evidence to date that there is no requirement for a positive charge in mu- and delta-opioid receptor antagonists. In addition, cyclic hexapeptide 4 lacks a phenolic hydroxyl group. Taken together, these data suggest that the prevailing assumptions about delta- and mu-opioid receptor binding need revision and that the receptors for these opioid ligands have much in common with the NK-1 and somatostatin receptors.
最近我们报道了利用微型文库来取代强效生长抑素激动剂L-363,301（c[-Pro-Phe-D-Trp-Lys-Thr-Phe-]，以生长抑素（SRIF）的编号方式，Lys为第9位氨基酸）中的Lys(9)，从而生成强效神经激肽受体（NK-1）拮抗剂c[-Pro-Phe-D-Trp-p-F-Phe-Thr-Phe-]。这种新型环六肽不与SRIF受体结合。因此，单一突变就将L-363,301（一种在实验动物中效力约为SRIF的2 - 8倍的SRIF激动剂(24)）转变为一种体外IC(50)为2 nM的选择性NK-1受体拮抗剂。在针对δ-阿片受体配体的同一文库筛选过程中，我们鉴定出了四种化合物（1 - 4），它们代表了一类新型的δ-阿片拮抗剂，其中一些也是NK-1受体拮抗剂。最有效的δ-阿片拮抗剂c[-Pro-1-Nal-D-Trp-Tyr-Thr-Phe-]（2）在小鼠输精管试验中显示K(e)值为128 nM，在大鼠脑膜结合试验中δ-受体结合亲和力常数为152 nM。这些结果令人关注，因为它们代表了一类新型的δ-阿片拮抗剂，并且与之前报道的两种δ-阿片拮抗剂一样，它们都不带正电荷。为了进一步研究δ-阿片配体中对正电荷的需求，我们制备了两种源自β-酪蛋白吗啡的混合μ-激动剂/δ-拮抗剂H-Dmt-c[-D-Orn-2-Nal-D-Pro-Gly-]（7）的类似物，在其中我们通过伯氨基的甲酰化（5）或删除该N端氨基（6）来消除正电荷。后一种化合物被证明是δ-阿片拮抗剂，K(e)值在16 - 120 nM范围内，同时也是相当有效的μ-阿片拮抗剂（K(e)约为200 nM）。这六种化合物提供了迄今为止最有说服力的证据，表明μ-和δ-阿片受体拮抗剂不需要正电荷。此外，环六肽4缺乏酚羟基。综上所述，这些数据表明关于δ-和μ-阿片受体结合的普遍假设需要修正，并且这些阿片配体的受体与NK-1和生长抑素受体有许多共同之处。
