Kopin Alan S, McBride Edward W, Chen Ci, Freidinger Roger M, Chen Duan, Zhao Chun-Mei, Beinborn Martin
Molecular Pharmacology Research Center, Department of Medicine, Tufts-New England Medical Center, 750 Washington Street, Box 7703, Boston, MA 02111, USA.
Proc Natl Acad Sci U S A. 2003 Apr 29;100(9):5525-30. doi: 10.1073/pnas.0831223100. Epub 2003 Apr 15.
The search for small-molecule drugs that act at peptide hormone receptors has resulted in the identification of a wide variety of antagonists. In contrast, the discovery of nonpeptide agonists has been far more elusive. We have used a constitutively active mutant of the cholecystokinin 2 receptor (CCK-2R) as a sensitive screen to detect ligand activity. Functional assessment of structural analogs of the prototype CCK-2R antagonist, L-365,260 [3R-N- (2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl)-N'-(3-methylphenyl)urea], resulted in the identification of a series of agonists. Each of the active molecules is an S enantiomer, whereas the corresponding R stereoisomers have little or no activity. Further in vitro and in vivo assessment at the wild-type receptor indicated that efficacy of the two most active ligands approached that of the endogenous hormone. The function of selected R and S enantiomers was differentially sensitive to a point mutation, N353L, within the putative CCK-2R ligand pocket. The results of this study highlight the potential of constitutively active receptors as drug screening tools and the interdependence of ligand stereochemistry and receptor conformation in defining drug efficacy.
寻找作用于肽激素受体的小分子药物已促成了多种拮抗剂的鉴定。相比之下，非肽激动剂的发现则要困难得多。我们使用了胆囊收缩素2受体（CCK-2R）的组成型活性突变体作为灵敏筛选方法来检测配体活性。对CCK-2R原型拮抗剂L-365,260 [3R-N-（2,3-二氢-1-甲基-2-氧代-5-苯基-1H-1,4-苯并二氮杂卓-3-基）-N'-（3-甲基苯基）脲]的结构类似物进行功能评估，结果鉴定出了一系列激动剂。每个活性分子均为S对映体，而相应的R立体异构体几乎没有活性或完全没有活性。在野生型受体上进一步的体外和体内评估表明，两种活性最高的配体的效力接近内源性激素的效力。所选R和S对映体的功能对假定的CCK-2R配体口袋内的点突变N353L具有不同的敏感性。本研究结果突出了组成型活性受体作为药物筛选工具的潜力以及配体立体化学和受体构象在确定药物效力方面的相互依赖性。
