SU5416 is a Ligand of the AHR
To confirm that this molecule is a direct ligand of the AHR and not working through some other agonist, we performed competitive binding assays of the AHR using a radioligand. Photoaffinity experiments incubating 125IBr2N3DpD with the hepatic cytosolic fraction from C57BL/6J mice (AHRb isoform) were conducted as described in the Methods [21]. Increasing concentrations of SU5416, TCDD, BNF, and 1,2-Benzanthracene (a ligand of low receptor affinity) were added. As shown in figure 2C, SU5416 competitively displaced the radiolabel with efficacy similar to TCDD.
Figure 2. Induction of DRE-mediated transcription by SU5416 is AHR dependent. A. The AHR-mutant C35 cell line was transfected with the AHRb, lacZ gene and a 36DRE-Luc construct. Controls were transfected with the empty pSPORT vector plus the reporter constructs. After 24 h, the cells were treated with 3 mM SU5416 or 0.3% (v/v) DMSO, then incubated for 18 more h. Induction of AHR activity was determined by normalizing the luciferase activity to b-galactosidase activity. White bars: Empty vector. Grey bars: AHR. Error bars: SD; (n = 3). B. Induction of DRE-mediated transcription by SU5416 is ARNT dependent. The ARNT-deficient C4 cell line was transfected with the human ARNT or the pSPORT parent vector. These cells were also cotransfected, treated and assayed as in A. White bars: Empty vector. Grey Bars: ARNT. Error bars: SD; (n = 3). C. SU5416 is a ligand of the AHR. The hepatic cytosolic fraction from C57BL/6J mice was incubated with 1 nM of the radioligand 125BR2N3DpD, in the presence of increasing concentrations of competitor, SU5416, TCDD, BNF or 1,2-Benzanthracene. Ordinate: Specifically bound radioligand in the presence of competitor divided by specifically bound radioligand in the absence of competitor. Abscissa: The concentration of competing ligand, represented as log of molar concentration. Each data point represents the average of two determinations. Competitive binding to the C57BL/6J
cytosol produced the IC50 values of SU5416 = 2.1 nM, TCDD = 1.5 nM, BNF = 2.8 nM, and 1,2-Benzanthracene = 13.7 nM.

In utero Exposure to SU5416 Stimulates Closure of DV
We have previously shown that genetically altered mice that express only 10% of the AHR display a patent ductus venosus (DV) in the liver in nearly all cases [22]. We additionally identified that in utero activation of the receptor in the hypomorphs with TCDD successfully closed the DV [5]. To test the role of SU5416 as an in vivo ligand and its potential effect on embryology and vascular development, we performed timed matings of female AHRfxneo/+ mice to male AHRfxneo/fxneo mice. The pregnant dams were treated at embryonic day E18.5 with a single dose of SU5416 at 110 mg/kg, or an equivalent volume of the vehicle, corn oil. At 4 weeks of age, the pups were sacrificed, and DV status was examined by hepatic perfusion with trypan blue. As seen in Table 1, only 1 of 25 AHRfxneo/fxneo pups treated with corn oil possessed a closed DV. In the experimental group, 13 of 22 animals of this phenotype exposed to SU5416 had a closed DV.activity indicating loss of binding to the DRE, which is clearly in contrast to the long duration DRE-binding seen with TCDD. Of note, when we did titrate SU5416 doses as high as 10 mM, we did observe as much as 20% of TCDD response (1 nM) as far out as 96 hours (data not shown). This SU5416 data is similar to the known plasma half-life of 30 minutes, although VEGF-receptor inhibitor effects have been shown to last as much as 72 hours in culture [23]. We further analyzed whether the AHR antagonist CH223191 could inhibit the ability of SU5416 to activate the DRE in 101L-hepatoma cells. It has previously been shown that this antagonist inhibits TCDD but not some of the other ligands of the AHR including some polycyclic aromatic hydrocarbons. We first performed a titration of the AHR antagonist in culture with either 1 nM TCDD or 100 nM SU5416. As can be seen in figure S2B, the effects of TCDD are inhibited whereas minimal inhibition is shown for SU5416. In figure S2C, we show a titration of SU5416 with only a small amount of inhibition of activity by the antagonist (10 mM).

SU5416-induced Upregulation of CYP1A1 is Similar in Murine AHRb and AHRd Splenocytes
As the above in vitro experiments were performed in cell lines, we next utilized AHRb (C57BL/6J) and AHRd congenic mice (on a C57BL/6J background). Spleens from these mice were harvested and suspended in culture media, and exposed to titrating doses of TCDD and SU5416. These data are presented in figure 4, where the graphs show normalized data from 0 to 100% response. Normalized data was chosen to allow comparison of CYP1A1 upregulation to its maximum in AHRb versus AHRd mice. After 4 hours of culture, TCDD induced CYP1A1 more rapidly and to a higher degree in wild-type than AHRd splenocytes, with an EC50 of 0.461 nM in wild-type and 1.894 nM in AHRd animals. Figure 4B shows that SU5416 induced CYP1a1 similarly in AHRb and AHRd mice, with an EC50 of 0.682 nM in wild-type and 0.730 nM in AHRd mice. Figures S3A and B show the total fold change seen by qPCR analysis of splenocytes after exposure to TCDD and SU5416, to allow an assessment of the potency of AHR activation of these two ligands with CYP1A1 induction as the readout. As can be seen in the figure, TCDD elicits more CYP1A1 in AHRb compared to AHRd mice, whereas SU5416 leads to the same or more CYP1A1 in AHRd mice. By this readout, TCDD and SU5416 have similar potency in AHRd cells, and TCDD is a stronger ligand in AHRb cells.

SU5416 Upregulates CYP1A1 and CYP1B1
The above data clearly shows that SU5416 is a ligand of the AHR. We now focused our attention on the strong response of SU5416 to the AHRd polymorphism in the screening assay, and compared the activity of this ligand in the high and low affinity polymorphisms. We utilized the wild type rat hepatoma cell line, 5L, which harbors the high affinity AHR isoform, and our newly created AHRd-15 cell line. As seen in figure 3A, we first performed a titration with TCDD and measured EROD activity. As expected, the activity of TCDD was shifted by 1.5 orders of magnitude to the left for the AHRb isoform. In contrast, when SU5416 was tested in vitro, the two curves virtually overlapped (figure 3B), showing equal potency for cytochrome P450 induction using the two cell lines. We also tested BNF, which as expected, showed a strong response with the 5L cell line and no response with the AHRd-15 cell line (figure 3C). As these experiments were done in cell lines, and in addition the AHRd-15 line combines a rat cell line with a transfected murine AHR, we further tested the ability of SU5416 to activate the AHR in vivo. Six-week old C57BL/6J mice (AHRb) and DBA/2J (AHRd) were orally administered 30, 80, or 120 mg of SU5416 per kg of body weight.