The Anti-Inflammatory Drug Leflunomide Is an Agonist of the Aryl Hydrocarbon Receptor

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxicity and biological activity of dioxins and related chemicals. The AhR influences a variety of processes involved in cellular growth and differentiation, and recent studies have suggested that the AhR is a potential target for immune-mediated diseases.During a screen for molecules that activate the AhR, leflunomide, an immunomodulatory drug presently used in the clinic for the treatment of rheumatoid arthritis, was identified as an AhR agonist. We aimed to determine whether any biological activity of leflunomide could be attributed to a previously unappreciated interaction with the AhR. The currently established mechanism of action of leflunomide involves its metabolism to A771726, possibly by cytochrome P450 enzymes, followed by inhibition of de novo pyrimidine biosynthesis by A771726. Our results demonstrate that leflunomide, but not its metabolite A771726, caused nuclear translocation of AhR into the nucleus and increased expression of AhR-responsive reporter genes and endogenous AhR target genes in an AhR-dependent manner. In silico Molecular Docking studies employing AhR ligand binding domain revealed favorable binding energy for leflunomide, but not for A771726. Further, leflunomide, but not A771726, inhibited in vivo epimorphic regeneration in a zebrafish model of tissue regeneration in an AhR-dependent manner. However, suppression of lymphocyte proliferation by leflunomide or A771726 was not dependent on AhR.These data reveal that leflunomide, an anti-inflammatory drug, is an agonist of the AhR. Our findings link AhR activation by leflunomide to inhibition of fin regeneration in zebrafish. Identification of alternative AhR agonists is a critical step in evaluating the AhR as a therapeutic target for the treatment of immune disorders

The Aryl Hydrocarbon Receptor Mediates Leflunomide- Induced Growth Inhibition of Melanoma Cells

A novel role of the dihydroorotatedehydrogenase (DHODH) inhibitor leflunomide as a potential anti-melanoma therapy was recently reported (Nature 471: 518-22, 2011). We previously reported that leflunomide strongly activates the transcriptional activity of the Aryl Hydrocarbon Receptor (AhR). We therefore tested whether the AhR regulates the anti-proliferative effects of leflunomide in melanoma. We first evaluated the expression of AhR in melanoma cells and found that AhR is highly expressed in A375 melanoma as well as in several other cancer cell types. To evaluate whether AhR plays a role in regulating the growth inhibitory effects of leflunomide in A375 cells, we generated a stable cell line from parental A375 cells expressing a doxycycline (DOX) inducible AhR shRNA. Using these cells in the absence or presence of DOX (normal AhR levels or AhR-knockdown, respectively) we found that the anti-proliferative effects of leflunomide, but not its metabolite A771726, were strongly dependent upon AhR expression. It has been well established that supplementation of cells with exogenous uridine completely rescues the anti-proliferative effects due to DHODH inhibition. Thus, we performed uridine rescue experiments in A375 cells to determine whether the anti-proliferative effects of leflunomide are solely due to DHODH inhibition as previously reported. Interestingly, saturating levels of uridine only modestly rescued A375 cells from the anti-proliferative effects of both leflunomide and A771726, indicating additional mechanism(s), apart from DHODH inhibition are responsible for the anti-proliferative effects of leflunomide in melanoma cells. Uridine also did not rescue MDA-MB-435S melanoma cell proliferation after leflunomide treatment. Our results reveal that the AhR is a molecular target of leflunomide and support the feasibility of the clinical application of leflunomide for treating melanoma. Furthermore, analysis of expression data from 967 cancer cell lines revealed that AhR is expressed in multiple different cancer types supporting the intriguing possibility of targeting the AhR for therapy in a number of cancers

Benzimidazoisoquinolines: a new class of rapidly metabolized aryl hydrocarbon receptor (AhR) ligands that induce AhR-dependent Tregs and prevent murine graft-versus-host disease

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

PunjSumitEnvironmentalMolecularToxicologyBenzimidazoisoquinolines_SupportingInformation.zip

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

FIGURE 4 from Identification and Characterization of a Small Molecule Bcl-2 Functional Converter

Interaction of BFC1108 with Bcl-2. A, Limited proteolysis of Bcl-2 loop domain in the presence of BFC1108. Purified GST-tagged Bcl-2 loop domain and GST only control were incubated with 50 µmol/L BFC1108. The proteolysis pattern of loop domain was determined at the indicated times upon coincubation with trypsin. B, BFC1108 stabilizes and increases melting temperature (Tm) of Bcl-2 full-length protein. Thermal unfolding of Bcl-2 full-length protein in the presence of BFC1108 was monitored by SYPRO Orange fluorescence. Z-tag and no protein control samples were included to confirm BFC1108 specific interaction with Bcl-2. Two-way ANOVA with Sidak multiple comparisons post hoc test, **, P P C, BFC1108 stabilizes and increases Bcl-2 loop domain melting temperature (Tm). Thermal unfolding of Bcl-2 loop domain in the presence of BFC1108 monitored by SYPRO Orange fluorescence. One-way ANOVA with Dunnett multiple comparisons post hoc test, ****, P D, MDA-MB-231/Bcl-2, H460 cells were exposed to BFC1108 at 10 µmol/L concentration for 48 hours in a medium containing 10% serum. Change in conformation of Bcl-2 was determined by using Bcl-2 BH3 antibody followed by flow cytometric analysis.</p

FIGURE 2 from Identification and Characterization of a Small Molecule Bcl-2 Functional Converter

BFC1108 suppresses viability of multiple cancer cell types. A, Bcl-2–selective inhibition of clonogenic survival of MDA-MB-231 cells. MDA-MB-231/Vector and MDA-MB-231/Bcl-2 cells were exposed to 10 µmol/L concentration in medium containing 10% FBS for 48 hours and the colony formation was determined after 2 weeks. B, Quantification of colony formation data shown in A. ***, P C, Bcl-2–dependent induction of apoptosis by BFC1108. MDA-MB-231 cells with high or low Bcl-2 expression were treated with 10 µmol/L BFC1108 in a medium containing 10% FBS for 48 hours and apoptosis was determined by annexin V staining. Data from one representative experiment is shown. Refer to Supplementary Fig. S1 for additional independent experiments. D, BFC1108 inhibits viability of breast cancer cells with Bcl-2 expression. The indicated breast cancer cells were treated for 48 hours, and viability was measured using CellTiter-Glo assay. Data were analyzed by two-way ANOVA and Dunnett multiple comparison post hoc test, **, P P 50). E, BFC1108 suppresses viability of multiple cancer cell types with minimal effects on MCF10A nontransformed breast epithelial cells. Cells were treated for 48 hours in 10% serum containing medium and cell viability was determined. ***, P < 0.001.</p

FIGURE 6 from Identification and Characterization of a Small Molecule Bcl-2 Functional Converter

Antitumor effects of BFC1108 in an orthotopic breast cancer model. A, BFC1108 inhibits growth of high Bcl-2–expressing MDA-MB-231 TNBC cells in vivo. A total of 106 MDA-MB-231/Bcl-2 cells were implanted in the mammary fat pad of NOD.SCID mice (n = 8 per group). Once palpable tumors were detected, mice were randomized and treated with vehicle or BFC1108 at 100 mg/kg twice a week by intraperitoneal route. Tumor measurements were made with digital calipers twice a week. *, P B, The body weights of mice treated with vehicle or BFC1108 in A were measured twice a week. C, Induction of apoptosis of tumor cells by BFC1108 in vivo. Immunofluorescence was performed on frozen tumor sections to determine apoptosis by TUNEL staining. D, BFC1108 induces conformation change of Bcl-2 in tumor tissues. Bcl-2 conformational change was detected by staining with Bcl-2 BH3 antibody. E, Bax activation by BFC1108. Activated Bax was detected using Bax 6A7 antibody in tumor tissues from animals treated with vehicle or BFC1108. F, Activation of caspase-3 by BFC1108. Activated caspase-3 was detected using cleaved caspase-3 antibody in tumor tissues from animals treated with vehicle or BFC1108.</p

Supplemental Figure 1 from Identification and Characterization of a Small Molecule Bcl-2 Functional Converter

Bcl-2 dependent apoptotic effects of BFC1108. Independent experimental replicates of data in Figure 2C are shown.</p

Supplemental Figure 2 from Identification and Characterization of a Small Molecule Bcl-2 Functional Converter

Bioluminescent imaging of data in Figure 7 for individual mouse treated with vehicle or BFC1108 is shown.</p

FIGURE 3 from Identification and Characterization of a Small Molecule Bcl-2 Functional Converter

BFC1108 activates intrinsic mitochondrial death pathway. A, BFC1108 suppresses the colony-forming ability of MEF cells in a Bcl-2–dependent manner. WT and Bcl-2−/− MEF cells were treated for 48 hours in a medium containing 10% FBS and the colony formation was determined after 2 weeks. B, Quantification of colony formation data shown in A. **, P C, Bax or Bak is required for BFC1108-induced suppression of viability. WT MEF, Bax Knockout (Bax−/− Bak+/+), Bak knockout (Bax+/+ Bak−/−) and double knockout (Bax−/− Bak−/−) MEF cells were treated with 1 µmol/L BFC1108 for 24 hours in 10% FBS medium and viability was assessed using CellTiter-Glo assay. **, P P D, BFC1108 decreases mitochondrial membrane potential of Bcl-2–expressing H460 lung cancer cells. JC-1 dye was used to stain live H460 cells that were treated with 10 µmol/L BFC1108 for 16 hours in 10% FBS containing medium. Images taken with FITC, and rhodamine filters were overlaid. Cells stained orange have intact mitochondrial outer membrane and the ones turning green have compromised outer membrane indicating loss of membrane potential.</p