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

Abstract

The aryl hydrocarbon receptor (AHR) is critically involved in several physiologic processes, including cancer progression and multiple immune system activities. We, and others, have hypothesized that AHR modulators represent an important new class of targeted therapeutics. Here, ligand shape-based virtual modeling techniques were used to identify novel AHR ligands on the basis of previously identified chemotypes. Four structurally unique compounds were identified. One lead compound, 2-((2-(5-bromofuran-2-yl)-4-oxo-4H-chromen-3-yl)oxy) acetamide (CB7993113), was further tested for its ability to block three AHR-dependent biologic activities: triple-negative breast cancer cell invasion or migration in vitro and AHR ligand-induced bone marrow toxicity in vivo. CB7993113 directly bound both murine and human AHR and inhibited polycyclic aromatic hydrocarbon (PAH)-and TCDD-induced reporter activity by 75% and 90% respectively. A novel homology model, comprehensive agonist and inhibitor titration experiments, and AHR localization studies were consistent with competitive antagonism and blockade of nuclear translocation as the primary mechanism of action. CB7993113 (IC 50 3.3 Â 10 27 M) effectively reduced invasion of human breast cancer cells in three-dimensional cultures and blocked tumor cell migration in two-dimensional cultures without significantly affecting cell viability or proliferation. Finally, CB7993113 effectively inhibited the bone marrow ablative effects of 7,12-dimethylbenz[a]anthracene in vivo, demonstrating drug absorption and tissue distribution leading to pharmacological efficacy. These experiments suggest that AHR antagonists such as CB7993113 may represent a new class of targeted therapeutics for immunomodulation and/or cancer therapy.

Role for the Aryl Hydrocarbon Receptor and Diverse Ligands in Oral Squamous Cell Carcinoma Migration and Tumorigenesis

“…To determine if the AHR in oral squamous carcinoma cells was functional, HSC-3 and CAL27 cells, which express significant AHR protein levels (Supplemental Figure S1), were transiently transfected with an AHR-dependent luciferase reporter plasmid (pGudLuc) [31], treated with various AHR ligands in the absence or presence of AHR inhibitors CH223191 [38] or CB7993113 [33] (10 μM), and luciferase activity assayed. The baseline level of AHR (reporter) activity in both HSC-3 (Figure 2A) and CAL27 (Figure 2B) cells significantly decreased following addition of CH223191 or CB7993113 (p<0.05-0.005), supporting the hypothesis that the AHR was “constitutively active”, presumably because of endogenous ligands in these lines.…”

“…Here, this model was used to further explore the role of the AHR in OSCC and to begin to assess if AHR inhibition represents a viable, targeted approach to OSCC therapy. Indeed, daily oral lavage with CB7993113, a non-toxic competitive AHR inhibitor [33], significantly reduced tumor load and weight loss and increased overall survival. The lack of weight loss observed in CB7993113-treated mice over the course of the experiment was consistent with the lack of toxicity seen with this potential therapeutic in vitro [33].…”

“…Indeed, daily oral lavage with CB7993113, a non-toxic competitive AHR inhibitor [33], significantly reduced tumor load and weight loss and increased overall survival. The lack of weight loss observed in CB7993113-treated mice over the course of the experiment was consistent with the lack of toxicity seen with this potential therapeutic in vitro [33]. Of note, studies from other laboratories demonstrate that AHR down-regulation blocks B(a)P-induced OSCC development by blocking DNA adduct formation, likely through inhibition of AHR-dependent CYP1A1 and CYP1B1 induction [50, 51].…”

“…To address the possible contribution of environmental and microbial AHR ligands to OSCC, several classes of AHR ligands were used including a genotoxic PAH (B(a)P), a non-genotoxic but persistent halogenated hydrocarbon (TCDD), PAH-containing 2.5 μm airborne particles (PM 2.5 ), and products from Pseudomonas aeruginosa and Porphyromonas gingivalis , the latter a primary etiological agent associated with chronic periodontitis. To assess the feasibility of targeting the AHR signaling pathway as a novel OSCC treatment, we used two OSCC cell lines, a unique OSCC orthotopic xenograft model [32], a novel AHR inhibitor (CB7993113) [33], and molecular knockdown techniques.…”

The Aryl Hydrocarbon Receptor is a Critical Regulator of Tissue Factor Stability and an Antithrombotic Target in Uremia

“…A minor nuclear fraction indicated basal activation. Both IS and uremic serum induced AHR nuclear translocation within 20 minutes (Figure 1, B and C), an effect abrogated by a novel AHR antagonist, CB7993113, developed by our group 18 and CH223191, a well established AHR antagonist 19 ( Figure 1C, Supplemental Figure 5). …”