Becker J A, Wallace A, Garzon A, Ingallinella P, Bianchi E, Cortese R, Simonin F, Kieffer B L, Pessi A
Ecole Supérieure de Biotechnologie de Strasbourg, 67400 Illkirch, France.
J Biol Chem. 1999 Sep 24;274(39):27513-22. doi: 10.1074/jbc.274.39.27513.
We have screened a synthetic peptide combinatorial library composed of 2 x 10(7) beta-turn-constrained peptides in binding assays on four structurally related receptors, the human opioid receptors mu, delta, and kappa and the opioid receptor-like ORL1. Sixty-six individual peptides were synthesized from the primary screening and tested in the four receptor binding assays. Three peptides composed essentially of unnatural amino acids were found to show high affinity for human kappa-opioid receptor. Investigation of their activity in agonist-promoted stimulation of [(35)S]guanosine 5'-3-O-(thio)triphosphate binding assay revealed that we have identified the first inverse agonist as well as peptidic antagonists for kappa-receptors. To fine-tune the potency and selectivity of these kappa-peptides we replaced their turn-forming template by other turn mimetic molecules. This "turn-scan" process allowed the discovery of compounds with modified selectivity and activity profiles. One peptide displayed comparable affinity and partial agonist activity toward all four receptors. Interestingly, another peptide showed selectivity for the ORL1 receptor and displayed antagonist activity at ORL1 and agonist activity at opioid receptors. In conclusion, we have identified peptides that represent an entirely new class of ligands for opioid and ORL1 receptors and exhibit novel pharmacological activity. This study demonstrates that conformationally constrained peptide combinatorial libraries are a rich source of ligands that are more suitable for the design of nonpeptidal drugs.
我们在针对四种结构相关受体（人μ、δ和κ阿片受体以及类阿片受体ORL1）的结合试验中，筛选了一个由2×10⁷个β-转角受限肽组成的合成肽组合文库。从初步筛选中合成了66个单独的肽，并在四种受体结合试验中进行了测试。发现三种主要由非天然氨基酸组成的肽对人κ阿片受体具有高亲和力。在激动剂促进的[(³⁵)S]鸟苷5'-3'-O-(硫代)三磷酸结合试验中对其活性进行研究发现，我们鉴定出了首个κ受体反向激动剂以及肽类拮抗剂。为了微调这些κ肽的效力和选择性，我们用其他转角模拟分子替换了它们的转角形成模板。这个“转角扫描”过程使得能够发现具有改变的选择性和活性谱的化合物。一种肽对所有四种受体表现出相当的亲和力和部分激动剂活性。有趣的是，另一种肽对ORL1受体具有选择性，在ORL1处表现出拮抗剂活性，而在阿片受体处表现出激动剂活性。总之，我们鉴定出了代表阿片受体和ORL1受体全新一类配体且具有新型药理活性的肽。这项研究表明，构象受限的肽组合文库是更适合非肽类药物设计的丰富配体来源。
