Comparative In Vitro and In Silico Analysis of the Selectivity of Indirubin as a Human Ah Receptor Agonist

Abstract

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that modulates gene expression following its binding and activation by structurally diverse chemicals. Species differences in AhR functionality have been observed, with the mouse AhR (mAhR) and human AhR (hAhR) exhibiting significant differences in ligand binding, coactivator recruitment, gene expression and response. While the AhR agonist indirubin (IR) is a more potent activator of hAhR-dependent gene expression than the prototypical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), it is a significantly less potent activator of the mAhR. DNA binding analysis confirmed the greater potency/efficacy of IR in stimulating transformation/DNA binding of the hAhR in vitro and domain-swapping experiments demonstrated that the enhanced response to IR was primarily due to the hAhR ligand binding domain (LBD). Site-directed mutagenesis and functional analysis studies revealed that mutation of H326 and A349 in the mAhR LBD to the corresponding residues in the hAhR LBD significantly increased the potency of IR. Since these mutations had no significant effect on ligand binding, these residues likely contribute to an enhanced efficiency of transformation/DNA binding by IR-bound hAhR. Molecular docking to mAhR LBD homology models further elucidated the different roles of the A375V mutation in TCDD and IR binding, as revealed by [3H]TCDD competitive binding results. These results demonstrate the differential binding of structurally diverse ligands within the LBD of a given AhR and confirm that amino acid differences within the LBD of AhRs contribute to significant species differences in ligand response.

A Biomimetic, One-Step Transformation of Simple Indolic Compounds to Malassezia-Related Alkaloids with High AhR Potency and Efficacy

“…According to this hypothesis, the synthesis of 6'-bromoindirubin (2h) and 3-bromotryptanthrin (3h) seems to be favored compared to Studies from several laboratories have demonstrated that indirubin is a highly potent and efficacious human-selective AhR agonist that induces AhR-dependent gene expression in human cells with an EC50 that is ~10-fold greater than that of TCDD. 3,34,35,76 In contrast, in mice or mouse cells, indirubin is ~100-less less potent than TCDD, but still highly efficacious. 34,35,76 Site-directed mutagenesis and functional analysis studies have identified specific amino acid residues within the mouse and human AhR ligand binding pockets that contributes to this differential specificity and responsiveness.…”

“…3,34,35,76 In contrast, in mice or mouse cells, indirubin is ~100-less less potent than TCDD, but still highly efficacious. 34,35,76 Site-directed mutagenesis and functional analysis studies have identified specific amino acid residues within the mouse and human AhR ligand binding pockets that contributes to this differential specificity and responsiveness. 35 The similarities in structual aspects of indirubins and tryptanthrins suggest that similar species differences in tryptanthrin ligand specificity may also exist.…”

“…These induction and inhibition responses suggest that 3-bromotryptanthrin (and perhaps other tryptanthrins) may represent a novel group of AhR agonists that interact with residues within the AhR ligand binding pocket in a manner distinctly different from TCDD, indirubin and other ligands characterized to date. 34,35,[73][74][75][76][77] However, further detailed QSAR and docking analysis of these analogues into both human and mouse AhR ligand binding sites generated by molecular modeling are needed to understand the molecular mechanisms responsible for the high affinity/potency of selected tryptanthrins and the dramatic species differences in ligand specificity.…”

“…22 Indirubins were tested only in the human cell line due to previous evidence for the high selectivity of indirubins for the human AhR as compared to the mouse AhR. 3,34,35 In contrast, tryptanthrin analogues were tested in both stably transfected human (HG2L7.5c1) and mouse (H1L7.5c3)hepatoma cell lines, not only to determine their relative potencies and efficacies, but also to evaluate whether these analogues also exhibited a greater selectivity for human AhR compared to mouse AhR. The 6 hour concentration-response results of tryptanthrins obtained using the human HG2L7.5c1 cells are presented in While most of the synthesized indirubins showed very strong activity (two of them (2b, 2f) had potencies comparable to or greater than TCDD), insertion of substituents reduced activity in comparison to the highly potent unsubstituted indirubin (Table 5).…”

“…interestingly showed a 350-fold increased potency compared to tryptanthrin (3) in the human HG2L7.5c1 cells and was comparable to the potency of TCDD when evaluated at 6h (EC50 5x10 -10 M. In contrast, the monosubstituted analogue 3h (3-bromotryptanthrin) was 3,000-times more potent than tryptanthrin and 10 times more potent than TCDD in the human cells. Given the previously documented high degree of selectivity of indirubin for human AhR, as compared to mouse AhR, 3,34,35 and similarities in several structural aspects of indirubin and tryptanthrin, the relative potencies of tryptanthrin analogues in human and mouse cells were compared. These analyses revealed that while most tryptanthrin analogues were about 10-fold less potent in the mouse cell line, as compared to that of the human cell line, all were significantly less potent than TCDD in the mouse cells ( Table 6, Figure 4).…”