To establish that GNF351 is a direct ligand for the AHR, a ligand competition binding assay using the PAL was performed

To establish that GNF351 is a direct ligand for the AHR, a ligand competition binding assay using the PAL was performed. in the footnote. After the discovery of this class of compounds, it was hypothesized that a class of AHR antagonists may exist, which not only inhibits the DRE response, but also fails to exhibit SAhRM activity. Though a number of AHR antagonists are Carboxin known and have been used in past studies, these compounds were characterized only in the context of antagonism of an agonist and thus may only antagonize DRE-mediated AHR activity. Also whether these AHR antagonists exhibit SAhRM activity remains to be explored. This article establishes that transcripts normalized to = 3/treatment group) and were analyzed to determine significance. Results GNF351 Is an AHR Ligand. A screen conducted to identify compounds with the capacity to expand CD34+ hematopoietic stem cells in vitro identified SR1, which elicits its activity Rabbit polyclonal to AADACL3 through antagonism of the AHR (Boitano et al., 2010). In fact, SR1 is a potent AHR antagonist that exhibits species selectivity in that it inhibits human AHR but not mouse or rat AHR. Medicinal chemistry optimization was used to synthesize GNF351, a closely related analog of SR1 that also displayed potent AHR antagonist activity. The structure of GNF351, as well as other AHR agonists and Carboxin antagonists used or discussed Carboxin in this study, are found in Fig. 1. To establish that GNF351 is a direct ligand for the AHR, a ligand competition binding assay using the PAL was performed. Figure 2 demonstrates that GNF351 is capable of competing with the photoaffinity ligand for binding to the human AHR and has a relative affinity for the AHR similar to that of SR1. These data demonstrate that GNF351 has a relatively high affinity for the receptor. Open in a separate window Fig. 1. Structures of the AHR antagonist GNF351 and other AHR ligands used in this study. Open in a separate window Fig. 2. GNF351 is an AHR ligand. A competition AHR ligand binding assay was conducted as described under levels, normalized to levels, was examined. Each treatment was conducted in triplicate wells. Data represent the mean S.E.M. with statistically significant results indicated (*, 0.05; **, 0.01; ***, 0.001), which are relevant to the data sets as labeled in A and compared with control for B. The direct comparisons are shown by the presence of the same letter (a or b). GNF351 Antagonizes Ligand-Mediated AHR Transcriptional Activity. HepG2 40/6 cells were treated with GNF351 in combination with TCDD for 4 h to determine whether GNF351 inhibits the potent agonist effect seen with TCDD treatment. As the concentration of GNF351 increased, the AHR DRE-mediated response was antagonized in a dose-dependent manner (Fig. 4A). To determine whether this effect is species-specific, H1L1.1.1c2 cells were also treated with increasing concentrations of GNF351 in combination with TCDD. Figure 4B shows that GNF351 antagonizes the agonist response in a mouse cell line in a dose-dependent manner, although it took a higher concentration of GNF351 to give the same antagonistic effect as that seen in the HepG2 40/6 cells. This was not unexpected, considering that the murine AHR has a 10-fold higher affinity for TCDD. Taking this into account it would seem that the affinity of GNF351 for the mouse and human AHR are similar. Quantitative PCR was conducted with HepG2 40/6 cells treated with vehicle, TCDD (2 nM), or a combination of GNF351 (100 nM) and TCDD (2 Carboxin nM) for 4 h. Figure 5 shows that levels of transcribed decreased with the combined treatment. These three genes have previously been shown to be AHR-responsive (Beischlag et al., 2008). The data further show that the antagonistic effect of GNF351 is not limited to one specific AHR-dependent gene. Considering that a 4-h treatment was able to decrease constitutive AHR target gene expression, it is likely that further repression would be observed with a longer GNF351 treatment. Open in a separate window Fig. 4. GNF351 antagonizes the DRE-mediated response in AHR in human and murine cells. Cells were treated with increasing concentrations of GNF351 in combination with 5 nM TCDD in stable human hepatoma-derived reporter cells (HepG2 40/6) (A) and with 2 nM TCDD in stable murine hepatoma-derived reporter cells (H1L1.1.1c2) (B) for 4 h, after which.