Carazo Alejandro, Pávek Petr
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University in Prague, Heyrovského 1203, Hradec Kralove CZ500 05, Czech Republic.
Sensors (Basel). 2015 Apr 21;15(4):9265-76. doi: 10.3390/s150409265.
The constitutive androstane receptor (CAR) is a critical nuclear receptor in the gene regulation of xenobiotic and endobiotic metabolism. The LanthaScreen(TM) TR-FRET CAR coactivator assay provides a simple and reliable method to analyze the affinity of a ligand to the human CAR ligand-binding domain (LBD) with no need to use cellular models. This in silico assay thus enables the study of direct CAR ligands and the ability to distinguish them from the indirect CAR activators that affect the receptor via the cell signaling-dependent phosphorylation of CAR in cells. For the current paper we characterized the pharmacodynamic interactions of three known CAR inverse agonists/antagonists-PK11195, clotrimazole and androstenol-with the prototype agonist CITCO (6-(4-chlorophenyl)imidazo[2,1-b][1,3] thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime) using the TR-FRET LanthaScreen(TM) assay. We have confirmed that all three compounds are inverse agonists of human CAR, with IC50 0.51, 0.005, and 0.35 μM, respectively. All the compounds also antagonize the CITCO-mediated activation of CAR, but only clotrimazole was capable to completely reverse the effect of CITCO in the tested concentrations. Thus this method allows identifying not only agonists, but also antagonists and inverse agonists for human CAR as well as to investigate the nature of the pharmacodynamic interactions of CAR ligands.
组成型雄烷受体（CAR）是外源性和内源性代谢基因调控中的关键核受体。LanthaScreen™ TR-FRET CAR共激活因子检测提供了一种简单可靠的方法，无需使用细胞模型即可分析配体与人CAR配体结合域（LBD）的亲和力。因此，这种计算机模拟检测能够研究直接的CAR配体，并能够将它们与通过细胞中CAR的细胞信号依赖性磷酸化影响受体的间接CAR激活剂区分开来。在本论文中，我们使用TR-FRET LanthaScreen™检测方法，对三种已知的CAR反向激动剂/拮抗剂——PK11195、克霉唑和雄烯醇——与原型激动剂CITCO（6-(4-氯苯基)咪唑[2,1-b][1,3]噻唑-5-甲醛-O-(3,4-二氯苄基)肟）之间的药效学相互作用进行了表征。我们已经证实，这三种化合物都是人CAR的反向激动剂，IC50分别为0.51、0.005和0.35 μM。所有化合物也都拮抗CITCO介导的CAR激活，但只有克霉唑在测试浓度下能够完全逆转CITCO的作用。因此，这种方法不仅可以识别激动剂，还可以识别针对人CAR的拮抗剂和反向激动剂，并能够研究CAR配体药效学相互作用的性质。
