Diazepam Promotes Translocation of Human Constitutive Androstane Receptor (CAR) via Direct Interaction with the Ligand-Binding Domain

Abstract

The constitutive androstane receptor (CAR) is the essential regulator of genes involved both in xenobiotic and endobiotic metabolism. Diazepam has been shown as a potent stimulator of CAR nuclear translocation and is assumed as an indirect CAR activator not interacting with the CAR cavity. In this study, we sought to determine if diazepam is a ligand directly interacting with the CAR ligand binding domain (LBD) and if it regulates its target genes in a therapeutically relevant concentration. We used different CAR constructs in translocation and luciferase reporter assays, recombinant CAR-LBD in a TR-FRET assay, and target genes induction studied in primary human hepatocytes (PHHs), HepaRG cells, and in CAR humanized mice. We also used in silico docking and CAR-LBD mutants to characterize the interaction of diazepam and its metabolites with the CAR cavity. Diazepam and its metabolites such as nordazepam, temazepam, and oxazepam are activators of CAR+Ala in translocation and two-hybrid assays and fit the CAR cavity in docking experiments. In gene reporter assays with CAR3 and in the TR-FRET assay, only diazepam significantly interacts with CAR-LBD. Diazepam also promotes up-regulation of CYP2B6 in PHHs and in HepaRG cells. However, in humanized CAR mice, diazepam significantly induces neither CYP2B6 nor Cyp2b10 genes nor does it regulate critical genes involved in glucose and lipids metabolism and liver proliferation. Thus, we demonstrate that diazepam interacts with human CAR-LBD as a weak ligand, but it does not significantly affect expression of tested CAR target genes in CAR humanized mice.

CYP2B6 allelic variants and non-genetic factors influence CYP2B6 enzyme function

“…In two subjects, one with CYP2B6*1/*1 , the other with CYP2B6*5/*6 genotype, and predicted to be ‘normal’ and ‘intermediate’ metabolizers, respectively, the low S- mephenytoin N -demethylation was attributed to the antihypertensive amlodipine therapy that might have transiently evoked poor CYP2B6 activity. Furthermore, the exposure to CYP2B6 inducers, including the antibiotics rifampicin, the corticosteroid derivative prednisolone, cortisone, hydrocortisone and dexamethasone, the benzodiazepine diazepam and midazolam, and the calcium channel blocker felodipine induces transcriptional expression of CYP2B6 gene via nuclear receptors (PXR, CAR) 25 , 50 , 52 , 53 , 55 , 57 , 71 . Rifampicin treatment has been demonstrated to substantially increase the clearance of bupropion and the formation of hydroxy-bupropion metabolite 72 .…”

Searching for Constitutive Androstane Receptor Modulators

“…The second approach relies on the use of natural (CAR3; Keminer et al, 2019;Skoda et al, 2020) or artificial CAR variants where the basal activity is reduced by addition of amino acids to the human (Chen et al, 2010;Imai et al, 2013;Kanno et al, 2010) or animal CAR LBDs (Omiecinski et al, 2011;Pinne et al, 2016). This approach carries the risk of affecting selectivity of the LBP (CAR2 or insertions near H12) or the CAR/RXR binding (CAR3 or insertion of single alanine instead of APYLT) that may affect the ligand-dependent changes in coactivator recruitment.…”

“…The dynamic range of the employed reporter assays, as defined by the maximal response of established positive controls such as TCPOBOP or CITCO relative to the DMSO vehicle, varies significantly. These values range from about two-fold (Yao et al, 2010;Wahlang et al, 2014;Hardesty et al, 2018), to ~5-fold (Mäkinen et al, 2003;Lynch et al, 2019) and to 21-fold (Küblbeck et al, 2011a) with CAR1, and between 6-to 26-fold with CAR3 (Skoda et al, 2020;Keminer et al, 2019). A low dynamic range may limit the sensitivity to detect true activators, and more importantly, observed lack of reporter activity in a suboptimal assay does not indicate discovery of an indirect CAR activator.…”

“…As mentioned earlier, the co-expression, common target genes and ligand sharing by CAR and PXR complicates identification of true human CAR modulators in primary hepatocytes or in vivo. Knockout and humanized CAR and PXR animals (Scheer and Wolf, 2014;Skoda et al, 2020) can assist in dissection and verification of CYP induction process. A similar and very promising approach is the generation of human HepaRG knockout cell lines which do not express CAR or PXR.…”

Critical evaluation of the human relevance of the mode of action for rodent liver tumor formation by activators of the constitutive androstane receptor (CAR)

“…lack of hepatocyte RDS data at known carcinogenic dose levels), a number of other substances also appear to produce liver tumors in the mouse and/or rat by a CAR activation MOA. Examples include chlordane (Khasawinah and Grutsch 1989;Ross et al 2010), dalcetrapib (Hoflack et al 2012), diazepam (de la Iglesia et al 1981;IARC 1996;Skoda et al 2020), and ginkgo biloba extract (Maeda et al 2015).…”