Benzimidazoisoquinolines: A New Class of Rapidly Metabolized Aryl Hydrocarbon Receptor (AhR) Ligands that Induce AhR-Dependent Tregs and Prevent Murine Graft-Versus-Host Disease

Abstract

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that plays multiple roles in regulation of immune and inflammatory responses. The ability of certain AhR ligands to induce regulatory T cells (Tregs) has generated interest in developing AhR ligands for therapeutic treatment of immune-mediated diseases. To this end, we designed a screen for novel Treg-inducing compounds based on our understanding of the mechanisms of Treg induction by the well-characterized immunosuppressive AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We screened a ChemBridge small molecule library and identified 10-chloro-7H-benzimidazo[2,1-a]benzo[de]Iso-quinolin-7-one (10-Cl-BBQ) as a potent AhR ligand that was rapidly metabolized and not cytotoxic to proliferating T cells. Like TCDD,10-Cl-BBQ altered donor CD4+ T cell differentiation during the early stages of a graft versus host (GVH) response resulting in expression of high levels of CD25, CTLA-4 and ICOS, as well as several genes associated with Treg function. The Treg phenotype required AhR expression in the donor CD4+ T cells. Foxp3 was not expressed in the AhR-induced Tregs implicating AhR as an independent transcription factor for Treg induction. Structure-activity studies showed that unsubstituted BBQ as well as 4, 11-dichloro-BBQ were capable of inducing AhR-Tregs. Other substitutions reduced activation of AhR. Daily treatment with 10-Cl-BBQ during the GVH response prevented development of GVH disease in an AhR-dependent manner with no overt toxicity. Together, our data provide strong support for development of select BBQs that activate the AhR to induce Tregs for treatment of immune-mediated diseases.

Activation of the Aryl Hydrocarbon Receptor by 10-Cl-BBQ Prevents Insulitis and Effector T Cell Development Independently of Foxp3+ Regulatory T Cells in Nonobese Diabetic Mice

“…The dose of 10-Cl-BBQ was calculated empirically by determining a concentration that would induce equivalent Cyp1a1 expression as a body burden of 25 μg/kg TCDD, the therapeutic dose in NOD mice. Because NOD mice express the low responder AhR d allele, they require a higher dose of 10-Cl-BBQ to achieve comparable activation of AhR when compared to AhR b expressing C57BL/6 mice (13). Oral treatment with 60 mg/kg 10-Cl-BBQ induced commensurate expression of Cyp1a1 as the therapeutic dose of TCDD in NOD mice (Figure 1B).…”

“…Additionally, the notoriety of TCDD as an environmental toxicant would likely limit its acceptance for pharmacologic uses. To identify other high-affinity AhR ligands that exhibit more favorable pharmacokinetic properties than TCDD, we used small-molecule screening to discover a novel immunosuppressive AhR ligand, 10-Cl-BBQ (13). This nontoxic compound was shown to have an in vivo half-life of approximately two hours but could be administered to mice in a repeated dosing scheme to maintain AhR activation over time, similar to the prolonged AhR activation induced by TCDD.…”

“…This nontoxic compound was shown to have an in vivo half-life of approximately two hours but could be administered to mice in a repeated dosing scheme to maintain AhR activation over time, similar to the prolonged AhR activation induced by TCDD. Using an acute-graft-vs-host model, we found that 10-Cl-BBQ, like TCDD, directly targeted the AhR in CD4 + T cells to induce an AhR-dependent Treg phenotype, suppress the allogeneic CTL response and ameliorate the symptoms of GVHD (13). …”

Aryl Hydrocarbon Receptor–Dependent Pathways in Immune Regulation

“…Specifically, in this study, a novel water-soluble derivative of the compound VAF347 (VAG539) was found to promote longterm graft acceptance and active tolerance in BALB/c mice that received a transplant of MHC-mismatched pancreatic islet allografts. Also in the search for novel potential AhR-activating molecules to be used in transplantation tolerance, Punj et al screened a ChemBridge small molecule library (ChemBridge Corp, San Diego, CA) and identified 10-chloro-7H-benzimidazo[2,1-a]benzo[de]isoquinolin-7-one (10-Cl-BBQ) as a novel potent AhR ligand (30). They found that daily treatments with 10-Cl-BBQ during the GVHD response resulted in increased expression of several genes associated with Treg function and prevented the development of GVHD in an AhR-dependent manner.…”

Sinomenine induces the generation of intestinal Treg cells and attenuates arthritis via activation of aryl hydrocarbon receptor

“…[19][20][21] To examine whether AhR participates in the induction of SIN on Treg cells, the potential cytotoxicity of SIN in EL-4 cells was tested and the optimal concentrations (0.1, 0.3, and 1 mM) were used in the following studies, whereas TCDD was used as a positive control. SIN induced the expression of CYP1A1 (target gene of AhR) at protein and mRNA levels in a concentration-dependent manner, and it also markedly promoted the CYP1A1 activity (Figures 3a-c).…”