Identification of Amino Acid Residues in the Ligand-Binding Domain of the Aryl Hydrocarbon Receptor Causing the Species-Specific Response to Omeprazole: Possible Determinants for Binding Putative Endogenous Ligands

Abstract

Omeprazole (OME) induces the expression of genes encoding drug-metabolizing enzymes, such as CYP1A1, via activation of the aryl hydrocarbon receptor (AhR) both in vivo and in vitro. However, the precise mechanism of OME-mediated AhR activation is still under investigation. While elucidating speciesspecific susceptibility to dioxin, we found that OME-mediated AhR activation was mammalian species specific. Moreover, we previously reported that OME has inhibitory activity toward CYP1A1 enzymes. From these observations, we speculated that OME-mediated AhR target gene transcription is due to AhR activation by increasing amounts of putative AhR ligands in serum by inhibition of CYP1A1 activity. We compared the amino acid sequences of OME-sensitive rabbit AhR and nonsensitive mouse AhR to identify the residues responsible for the species-specific response. Chimeric AhRs were constructed by exchanging domains between mouse and rabbit AhRs to define the region required for the response to OME. OME-mediated transactivation was observed only with the chimeric AhR that included the ligandbinding domain (LBD) of the rabbit AhR. Site-directed mutagenesis revealed three amino acids (M328, T353, and F367) in the rabbit AhR that were responsible for OME-mediated transactivation. Replacing these residues with those of the mouse AhR abolished the response of the rabbit AhR. In contrast, substitutions of these amino acids with those of the rabbit AhR altered nonsensitive mouse AhR to become sensitive to OME. These results suggest that OME-mediated AhR activation requires a specific structure within LBD that is probably essential for binding with enigmatic endogenous ligands.

Microbial Metabolites of Omeprazole Activate Murine Aryl Hydrocarbon Receptor In Vitro and In Vivo

“…1B). These data agree with the results of previous studies (Dzeletovic et al, 1997;Shiizaki et al, 2014) and suggest that OME-induced mouse AhR activation depends on the cells in which AhR is expressed.…”

“…In our recent study, however, OME-mediated AhR activation was dependent on the AhR species. In particular, mouse AhR did not respond to OME even when it was expressed in human cells (Shiizaki et al, 2014). Although there seems to be a contradiction between these two insights, the present findings that an AhR ligand metabolically generated by yeast activates mouse AhR expressed in yeast might explain such conflicts without discrepancy.…”

“…In contrast to these observations, we recently found that OMEmediated transcriptional activation of the XRE-driven reporter gene was dependent on the species from which AhR originated but independent of the host cell species where AhR was expressed (Shiizaki et al, 2014). Specifically, human AhR introduced into murine hepatoma cells was activated by OME, whereas mouse AhR showed no response to OME, although it was expressed in human cells.…”

“…Plasmids used in this study were constructed and described in our previous report (Shiizaki et al, 2014). We used the reporter plasmid pX4TK-Luc that carries the firefly luciferase gene under the control of four copies of XRE and the thymidine kinase promoter.…”

Using Molecular Initiating Events to Develop a Structural Alert Based Screening Workflow for Nuclear Receptor Ligands Associated with Hepatic Steatosis

“…It was found that ligands must form interactions (usually via hydrogen bonds) with the key residues Met328, Tyr353 and Phe367 found within the AHR binding pocket in order to activate the AHR NR. 35 These structural features were then coded into SMARTS strings. 31 The AHR workflow was split into two parts, firstly the chemical must contain at least one of the backbone ring structures as reported in Table 4 (showing the SMARTS strings and visual representations) as these were observed as being essential to fitting into the binding pocket.…”