Identification of a High-Affinity Ligand That Exhibits Complete Aryl Hydrocarbon Receptor Antagonism

Abstract

The biological functions of the aryl hydrocarbon receptor (AHR) can be delineated into dioxin response element (DRE)-dependent or -independent activities. Ligands exhibiting either full or partial agonist activity, e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin and ␣-naphthoflavone, have been demonstrated to potentiate both DRE-dependent and -independent AHR function. In contrast, the recently identified selective AHR modulators (SAhRMs), e.g., 1-allyl-3-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-1H-indazole (SGA360), bias AHR toward DRE-independent functionality while displaying antagonism with regard to ligand-induced DRE-dependent transcription. Recent studies have expanded the physiological role of AHR to include modulation of hematopoietic progenitor expansion and immunoregulation. It remains to be established whether such physiological roles are mediated through DRE-dependent or -independent pathways. Here, we present evidence for a third class of AHR ligand, "pure" or complete antagonists with the capacity to suppress both DRE-dependent and -independent AHR functions, which may facilitate dissection of physiological AHR function with regard to DRE or non-DRE-mediated signaling. Competitive ligand binding assays together with in silico modeling identify N- (2-(1H-indol-3-yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine (GNF351) as a high-affinity AHR ligand. DRE-dependent reporter assays, in conjunction with quantitative polymerase chain reaction analysis of AHR targets, reveal GNF351 as a potent AHR antagonist that demonstrates efficacy in the nanomolar range. Furthermore, unlike many currently used AHR antagonists, e.g., ␣-naphthoflavone, GNF351 is devoid of partial agonist potential. It is noteworthy that in a model of AHR-mediated DRE-independent function, i.e., suppression of cytokine-induced acute-phase gene expression, GNF351 has the capacity to antagonize agonist and SAhRM-mediated suppression of SAA1. Such data indicate that GNF351 is a pure antagonist with the capacity to inhibit both DRE-dependent and -independent activity.

In Silico Identification of an Aryl Hydrocarbon Receptor Antagonist with Biological Activity In Vitro and In Vivo

“…1, arrows). The structures of the putative antagonist compounds and, for comparison, those of other previously described AHR antagonists, CH223191 (Kim et al, 2006), StemRegenin (SR1) (Boitano et al, 2010), GNF351 (Smith et al, 2011), and 6, 29,49trimethoxyflavone (Zhao et al, 2010) are provided in Fig. 2.…”

“…These include CH223191 (Kim et al, 2006), 6,29,49-trimethoxyflavone (TMF) (Murray IA et al, 2009), SR1 (Boitano et al, 2010), and GNF351 (Smith et al, 2011). Identification of additional AHR antagonists, such as CB7993113, is important because each antagonist appears to exhibit unique properties in terms of affinity for AHRs from different species, "ligand selectivity," or ability to block AHR response elementdependent signaling (Smith et al, 2011). For example, SR1 is a potent antagonist of the human AHR but has little or no effect on ligand binding to murine AHR.…”

The Aryl Hydrocarbon Receptor in Barrier Organ Physiology, Immunology, and Toxicology

“…Among the highaffinity exogenous substances are several planar and lipophilic PHAHs of the dioxin, dibenzofuran, biphenyl, and azoxybenzene types sharing structural properties with the highly toxic TCDD (Nguyen and Bradfield, 2008 (Till et al, 1999;Nguyen and Bradfield, 2008). The bacterial pigment 1-hydroxyphenazine and three pharmacological agents, StemRegenin 1 and GNF351 [N-(2-(3H-indol-3-yl)ethyl)-9-isopropyl-2-(5-methyl-3-pyridyl)-7H-purin-6-amine] (two potent AhR antagonists), as well as VAF347 [(4-(3-chloro-phenyl)-pyrimidin-2-yl)-(4-trifluoromethyl-phenyl)-amine], have also been observed to be high-affinity ligands (Lawrence et al, 2008;Boitano et al, 2010;Smith et al, 2011;MouraAlves et al, 2014). Many of these exogenous compounds probably bear structural similarities to physiologic ligand(s).…”

Hepatic Aryl Hydrocarbon Receptor Attenuates Fibroblast Growth Factor 21 Expression

“…3E). To confirm that the repressive action of AHR agonists occurs specifically through AHR, we attempted to suppress agonist-mediated activity by pretreating cells (16). Surprisingly, GNF351 was unable to antagonize 100 nM ICZ-mediated AHR activity (Fig.…”