Identification and Characterization of CINPA1 Metabolites Facilitates Structure-Activity Studies of the Constitutive Androstane Receptor

Abstract

The constitutive androstane receptor (CAR) regulates the expression of genes involved in drug metabolism and other processes. A specific inhibitor of CAR is critical for modulating constitutive CAR activity. We recently described a specific small-molecule inhibitor of CAR, CINPA1 (ethyl (5-(diethylglycyl)-10,11-dihydro-5H-dibenzo[b,f]azepin-3-yl)carbamate), which is capable of reducing CAR-mediated transcription by changing the coregulator recruitment pattern and reducing CAR occupancy at the promoter regions of its target genes. In this study, we showed that CINPA1 is converted to two main metabolites in human liver microsomes. By using cell-based reporter gene and biochemical coregulator recruitment assays, we showed that although metabolite 1 was very weak in inhibiting CAR function and disrupting CAR-coactivator interaction, metabolite 2 was inactive in this regard. Docking studies using the CAR ligand-binding domain structure showed that although CINPA1 and metabolite 1 can bind in the CAR ligand-binding pocket, metabolite 2 may be incapable of the molecular interactions required for binding. These results indicate that the metabolites of CINPA1 may not interfere with the action of CINPA1. We also used in vitro enzyme assays to identify the cytochrome P450 enzymes responsible for metabolizing CINPA1 in human liver microsomes and showed that CINPA1 was first converted to metabolite 1 by CYP3A4 and then further metabolized by CYP2D6 to metabolite 2. Identification and characterization of the metabolites of CINPA1 enabled structureactivity relationship studies of this family of small molecules and provided information to guide in vivo pharmacological studies.

Molecular Basis of Metabolism-Mediated Conversion of PK11195 from an Antagonist to an Agonist of the Constitutive Androstane Receptor

“…Therefore, PK11195 interactions with these residues may destabilize the AF2 domain, while loss of the N-methyl group in ND-PK could restabilize H12. Indeed, the potent CAR antagonist CINPA1 also interacts with these residues, suggesting such interactions are important in CAR antagonism (Cherian et al, 2016). CITCO interacts with V199, Y224, and Y326, but the interaction distances are generally greater than those of PK11195 or CINPA1 and thus may not displace these residues enough to inhibit stabilization of H12 (Supplemental Table 1).…”

“…8, B and C); notably, PK11195 and the CAR antagonist CINPA1 (Fig. 8D) share these interactions, which may contribute to their antagonism of hCAR (Cherian et al, 2016). The demethylation of PK11195 allows it move away and not interact with these important residues in the CAR binding pocket, which may explain how the difference of one methyl group between PK11195 and ND-PK can have such a substantial effect on CAR activity.…”

SPA70 is a potent antagonist of human pregnane X receptor

“…Docking of SPA70 to the hPXR LBD was conducted in a similar manner to that described elsewhere 58 and was performed using AutoDock Vina version 1.1.1 59 . All water and ligand molecules were removed from the crystal structure by using the Pymol molecular graphics system ( http://www.pymol.org ) before the protein and ligand PDBQT files needed for docking were generated using AutoDockTools (ADT) version 1.5.6 ( http://mgltools.scripps.edu/ ).…”

Sítios de interação alternativos em receptores nucleares e sua viabilidade como alvos terapêuticos usando triagem computacional e experimental.

“…The system allows multiplexing, which means that a single well can be used to determine a parameter for receptor activation, cell viability and induced enzyme activity [168][169][170]. Results from stable transfected cells can vary from standard transiently transfected assays and even primary cell-lines, which have to be taken into consideration [171,172].…”

“…Regardless of the new molecular study tools for CAR and the emergence value of this receptor as a drug target for several syndromes, so far only few inverse agonists are known: clotrimazole, meclizine, androstenol, PK11195 [96,118,122,124] and more recently CINPA1 and its analogues [125,126]. All compounds share at least some degree of cross-reactivity with other receptors, mainly PXR (Chai et al, 2016).…”