CINPA1 binds directly to constitutive androstane receptor and inhibits its activity

Abstract

The constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are xenobiotic sensors that regulate the expression of drug-metabolizing enzymes and efflux transporters. CAR activation promotes drug elimination, thereby reducing therapeutic effectiveness, or causes adverse drug effects via toxic metabolites. CAR inhibitors could be used to attenuate these adverse drug effects. CAR and PXR share ligands and target genes, confounding the understanding of the regulation of receptor-specific activity. We previously identified a small-molecule inhibitor, CINPA1, that inhibits CAR (without activating PXR at lower concentrations) by altering CAR-coregulator interactions and reducing CAR recruitment to DNA response elements of regulated genes. However, solid evidence was not presented for the direct binding of CINPA1 to CAR. In this study, we demonstrate direct interaction of CINPA1 with the CAR ligand-binding domain (CAR-LBD) and identify key residues involved in such interactions through a combination of biophysical and computational methods. We found that CINPA1 resides in the ligand-binding pocket to stabilize the CAR-LBD in a more rigid, less fluid state. Molecular dynamics simulations, together with our previously reported docking model, enabled us to predict which CAR residues were critical for interactions with CINPA1. The importance of these residues for CINPA1 binding were then validated by directed mutations and testing the mutant CAR proteins in transcription reporter and coregulatory interaction assays. We demonstrated strong hydrogen bonding of CINPA1 with N165 and H203 and identified other residues involved in hydrophobic contacts with CINPA1. Overall, our data confirm that CINPA1 directly binds to CAR.

The constitutive androstane receptor and pregnane X receptor in the brain

“…Ketoconazole Ketoconazole, L-sulforaphane, coumestrol, SPA70 (Cherian et al, 2018;Forman et al, 1998;Huang et al, 2007;Lemmen, Tozakidis, Bele, & Galla, 2013;Lin et al, 2017;Wang et al, 2008;Yeung, Sueyoshi, Negishi, & Chang, 2008) Biological (Aleksunes & Klaassen, 2012;Chen, Ferguson, Negishi, & Goldstein, 2003;Ferguson, Chen, LeCluyse, Negishi, & Goldstein, 2005;Ferguson, LeCluyse, Negishi, & Goldstein, 2002;Goodwin, Hodgson, D'Costa, Robertson, & Liddle, 2002;Maglich et al, 2004;Xu, Wang, & Staudinger, 2009;Yoshinari, Yoda, Toriyabe, & Yamazoe, 2010;Zhang, Huang, Chua, Wei, & Moore, 2002) UGTs Ugt1a1, Ugt1a9, Ugt2b34, Ugt2b35, Ugt2b36 UGT1A1 Ugt1a1, Ugt1a5, Ugt1a9 UGT1A1, UGT1A6, UGT1A3, UGT1A4 (Aleksunes & Klaassen, 2012;Maglich et al, 2004;…”

Strategies for developing pregnane X receptor antagonists: Implications from metabolism to cancer

“…This is because deleting the PXR gene not only abrogates its ligand‐dependent transcriptional activity but also abolishes the nonligand‐mediated roles of PXR, such as those relating to protein‐protein interactions. Such modulators should possess great selectivity to avoid off‐target effects; otherwise, their use may confound experimental results, leading to contradictory or puzzling conclusions, especially when PXR shares common target genes with the constitutive androstane receptor …”

Breach of ligand-receptor interaction in pathogenesis of disadaptive disorders and in pathology

“…По-видимому, здесь происходит прямое взаимодействие CINPA1 с лиганд-связывающим доменом CAR (CAR-LBD). И все же можно констатировать, что CINPA1 находится в лигандсвязывающем кармане для стабилизации CAR-LBD в более жестком, менее текучем состоянии при образовании сильной водородной связи CINPA1 и гидрофобных контактов [40].…”