Secondary Screen for Agonists of the Murine AHRd Low Affinity Receptor
The 100 “hit compounds” from the primary screen were subsequently screened for their capacity to activate the low-affinityFigure 1. Screen of small molecule library for AHR agonists. A. A collection of 4160 compounds was screened for the induction of the DRE-driven luciferase in the human hepatoma 101L cell line. In 384-well plates, 100 mL media containing 70% confluent 101L cells was incubated with 10 mM of each test compound (1% v/v DMSO) for 24 hours. Dotted line indicates 3-fold induction. B. Screen for agonists of the AHRd. The AHRd-15 cell line was treated with 1 mM of the 98 compounds identified from the primary screen, 2 nM TCDD or DMSO and EROD activity was determined. Dashed line indicates 5-fold induction.
murine AHRd receptor isoform using the activity of the endogenous Cyp1a1 gene as a readout. To this end, we established a hepatoma cell line that expresses the AHRd receptor isoform derived from the DBA/2J mouse [14]. An AHRd-expressing cell line was generated by stably transfecting the AHRd cDNA into the rat hepatoma AHR-deficient cell line, BP8 [15]. After stable selection with G418, a subclone (AHRd-15) was analyzed for receptor expression and function. First, a western blot using an anti-AHR antibody, revealed that the AHRd-15 cells produced an immunoreactive protein band that co-migrated with a receptor species isolated from the hepatic cytosol of DBA/2J mice (approximate size 104 kDa). This band was distinct from the AHRb isoform found in C57BL/6J cytosol, which migrated at 97 kDa (figure S1A). To confirm that the AHRd-15 clone expressed a functional low affinity AHRd isoform, we examined the receptor-mediated response to the prototype agonists, TCDD and b-naphthoflavone (BNF). Increasing concentrations of TCDD induced CYP1A1-mediated EROD activity in these cells with an EC50 in the 30 nM range [16]. In contrast, the much weaker agonist, BNF, known not to induce an AHR-mediated response in the AHRd receptor isoform expressed in the hepatocytes of DBA/ 2J mice [17], was shown to be inactive at doses as high as 10 mM in the AHRd-15 cells (figure S1B). To test the ability of the 100 AHR inducers to activate the AHRd-15 cells, they were treated with each of the compounds at the dose of 1 mM, for 36 hours in 96-well plates. Only the compound SU5416, and the positive control, TCDD, induced AHRd-mediated EROD activity greater than 5-fold (figure 1B). Therefore, SU5416 was considered for further analysis.
Induction of DRE-mediated Transcription by SU5416 is AHR and ARNT Dependent
To prove that induction of the DRE was mediated through classic AHR signal transduction, and not through a VEGF-related mechanism, we employed mutant cell lines that lack expression of the AHR or ARNT. The C35 cell line, which contains a dysfunctional AHR, was utilized [18]. It was transfected with vector containing the murine AHR gene, the lacZ gene, and the luciferase reporter gene driven by 3 upstream DREs, as described in the Methods section. Controls were mock transfected with reporter plasmids and the empty vector. Cells were treated with either 3 mM SU5416 or DMSO (control). As seen in figure 2A, cells transfected with the AHR plasmid generated significant luciferase activity when exposed to SU5416 compared to DMSO. The control cells generated minimal activity. In a similar experiment, the ARNT-deficient mouse hepatoma cell line C4 was transiently transfected with plasmids encoding human ARNT, the lacZ gene, and the same DRE-driven luciferase gene, and control samples received empty vectors for ARNT [19,20]. As shown in figure 2B, after exposure to SU5416 or DMSO, activity was only seen when ARNT was transfected.
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).
