At the request of one of my former postdocs (who is now a dept chair at Yale) — and who has been on this GEITP mailing list since its beginning in 2008 — today’s blog discusses an invited review that was just published in Nature Reviews Drug Discovery [see attached]. Authors state that “AHR” was named in 1976, but, in fact, it was named in 1974. This review summarizes beautifully the incredible advances during 51 years of AHR research.
Prologue: starting in 1967, a skinny postdoc (Nebert) in the Gelboin lab at the National Cancer Institute (NCI), NIH (Bethesda MD) was fascinated with early publications of Lee W Wattenberg (Univ Minnesota) and Alan H Conney (McArdle Inst, Univ Wisconsin, Madison) — showing that polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BaP, a chemical carcinogen found in cigarette smoke), when administered to rats, induce metabolism of the incoming BaP inducer. Nebert decided to develop a simple, reliable, quick assay to measure aryl hydrocarbon hydroxylase (AHH) activity in cell culture as well as in lab animals [JBC 1968; 243: 6242—cited more than 2,000 times]. This enzyme induction process became clinically more relevant when Nebert with Gelboin found AHH activity in placenta of cigarette smokers but not in nonsmokers [Cancer Res 1969; 29: 1763].
Moving to the National Institute of Child Health and Human Development (NICHD) in late 1968, Nebert first showed that “induced AHH enzyme activity” represented one or more cytochromes P450 (JBC 1970; 245: 519—now known to represent CYP1A1,CYP1A2,CYP1B1). The Nebert lab then showed that some inbred mouse strains (e.g., B6) were ‘highly responsive’ to PAH-induced AHH activity, whereas other mouse strains (e.g., D2) were not; ‘AHH induction responsiveness’ was inherited as a Mendelian dominant trait, ‘AHH induction non-responsiveness’ thus was an autosomal recessive trait (JBC 1972; 247: 1125). This meant that crossing a B6D2F1 mouse with an autosomal recessive D2 mouse (Fed Proc 1972; 31: 1315) — would produce a 50:50 distribution in utero of ‘AHH-induction responsive’ versus ‘AHH-induction nonresponsive’ embryos [thus, effects of CYP1-associated drugs could be studied transplacentally in utero where all animals experienced the same dose of drug — as opposed to the “usual control experiments” in which “controls” are simply not given any drug].
In 1974, Nebert saw a publication by Alan Poland & Edward Glover (Mol Pharmacol 1974; 10: 349) stating that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) was about 30,000 times more potent than the PAH 3-methylcholanthrene (3MC) at inducing chick embryo AHH activity. Nebert telephoned Poland the same day and invited him to visit the Nebert lab, where they confirmed that a ~20-times larger dose of TCDD in AHH-induction nonresponsive mice was able to “force” AHH induction to the same level as that in AHH-induction responsive mice. This dose-response curve finding is consistent with a structural gene encoding the AHH enzyme that is being controlled by a regulatory gene which we named aryl hydrocarbon receptor (AHR); the Ahr gene in genetically sensitive mice such as B6 would carry a high-affinity (b) allele, and genetically resistant mice such as D2 would carry a poor-affinity (d) allele (JBC 1974; 249: 5599 & Arch Biochem Biophys 1975; 166: 559).
In the Poland lab (JBC 1976; 251: 4936), following treatment with radiolabeled TCDD, liver cytosol accumulation of radiolabel was greatest in B6 mice, intermediate in B6D2F1 mice, and lowest in D2 mice. In the Nebert lab (Cell 1982; 31: 275), movement of radiolabeled TCDD from liver cytosol to the nucleus of AHH-induction responsive mice was highly correlated with Cyp1a1 messenger RNA (mRNA) induction kinetics; radioactivity in liver cytosol and nucleus of AHH-induction nonresponsive mice was 4- to 5-fold lower. These experiments provided direct proof that AHR is first bound to TCDD in cytosol, and, following migration to the nucleus, the ligand-bound AHR signal then initiates the enzyme induction response.
Respecting evolution, Nebert was convinced that AHR had not evolved simply to bind PAHs and TCDD (i.e., AHR must have arisen for one or more endogenous functions). In 1979, the Leland Chung lab (Boulder, CO) reported that AHH induction by 3MC was negligible in “ethanol Long-Sleep” (LS) mice, whereas AHH was highly inducible by 3MC in “ethanol Short-Sleep (SS) mice (JPET 1979; 209: 404). The “ethanol sleep time”, developed by pharmacologists, is essentially a test to see how rapidly rodents can recover from ethanol-induced peritonitis. Did this trait reflect differences in AHR affinity? The Nebert lab found that AHH-induction nonresponsive mice, like D2, acted like LS mice, and AHH-induction responsive mice, like B6, acted like SS mice [Annu Rev Pharmacol Toxicol 1981; 21: 431 & Biochem Pharmacol 1989; 38: 3565 & EXS 1994; 71: 231-40 (Nobel Symposium)]. In conclusion, AHR, in addition to responding to PAHs and TCDD, was somehow involved in the body’s pro-inflammatory and/or inflammation resolution phase. The Nebert lab proceeded to show that statistically significant changes in levels of leukotriene B4 and seven other lipid mediator (LM) metabolites are associated with AHR/CYP1-mediated proinflammatory and inflammation-resolution pathways caused by zymosan-initiated peritonitis (J Immunol 2013; 191: 3347).
Today, AHR/CYP1 signaling pathways and networks have been shown in dozens of labs to participate in over one hundred critical-life processes in more than a dozen organs and cell-types (reviewed in Progr Lipid Res 2017; 67: 38 & Front Toxicol 2021; 3: 787360 & Annu Rev Pharmacol Toxicol 2024; 64: 1).
Can AHR act as a druggable target to treat specific clinical disorders? The rational design of therapeutic small molecules that activate or inhibit AHR activity requires a detailed structural understanding of its interactions with many varied ligands. For example, characterizing the binding modes of endogenous AHR agonists provides a template for designing ligands with desired pharmacological properties — such as AHR modulators that selectively target specific biological functions.
Cryo electron microscopy structures of AHR–HSP90–XAP2 cytosolic complexes bound to the endogenous AHR agonist indirubin — revealed a large ligand-binding cavity that enables interaction with ligands of different sizes. These studies defined a preferential interaction of AHR with planar compounds, as well as the potential for ligands to differentially interact with residues in the binding pocket, resulting in conformational changes that selectively affect the interaction of the AHR with individual binding partners. These observations provide important structural insights into the reported ligand-dependent biological effects of AHR activation, and should assist drug developers in refining structure–activity relationships and optimizing drug candidates by using structure-based drug design and virtual screening.
Based on the structural work of AHR bound to its ligands, the rational design of selective AHR modulators (SAHRMs) is a promising approach to target specific AHR functions while minimizing off-target effects. In this context, although structural information is available on specific AHR domains, an important limitation is the lack of high-quality structural data on the full-length AHR protein.
Table 1 [see attached review] is fantastically complete in that it lists ~44 AHR ligands that include dioxin-like structures, tryptophan metabolites, indole metabolites, dietary chemicals, heme-derived metabolites, phytochemicals, and at least five arachidonic acid-derived (lipid mediator-derived) metabolites.
The canonical (the standard or accepted version) AHR-signaling pathway is initiated by agonist binding to the AHR–chaperone complex in the cytoplasm (see Fig. 1a of attached). This interaction induces conformational changes of AHR — which result in dissociation of AIP from the AHR complex and exposure of the N-terminal nuclear localization signal. The AHR–agonist complex then interacts with transportin and importin-β, which mediate its nuclear import.
Non-canonical (anything that deviates from standard) AHR-signaling transduction involves nuclear and/or cytosolic pathways (see Fig. 1b of attached). In nuclear mechanisms, the AHR–agonist complex interacts with and regulates activity of other transcription factors (e.g., AHR controls nuclear factor-κB (NFκB) activity through both direct interactions and indirect mechanisms; AHR directly interacts with RelA and RelB of the NFκB complex and interferes with transcriptional programs). Moreover, AHR nuclear translocation induces suppressor of cytokine-signaling-2 (SOCS2), which inhibits TLR-signaling and NFκB-dependent expression of cytokines — such as IL6, IL12a, IL12b, IL23a and TNF, thereby indirectly interfering with NFκB mechanisms. These direct and indirect mechanisms control NFκB-driven transcriptional programs in multiple cell types.
Finally, it has been proposed that AHR activation can be triggered in the absence of agonists (e.g., shear stress and oxidative stress were shown to induce AHR nuclear translocation and Cyp1a1 expression)…!! However, it is crucial to explore the significance of this ligand-independent AHR activation in a physiological setting.
Authors went on to summarize AHR functions in adaptive and innate immune responses (dendritic cells, innate lymphoid cells, CD4+ T lymphocytes, and other lymphocyte types). This was followed by up-to-date knowledge on “targeting AHR in inflammatory disorders” — starting off with inflammatory skin diseases. Authors summarized the latest on tapinarof (a naturally-occurring stilbene compound with AHR agonist activity) that was developed by Dermavant Sciences and approved by the FDA in 2022 for the treatment of plaque psoriasis in adults.
Other examples of targeting AHR in inflammatory disorders — include multiple sclerosis and experimental autoimmune encephalomyelitis, systemic lupus erythematosus, inflammatory bowel disease, asthma, and chronic obstructive pulmonary disease (COPD), infectious diseases, and cancer. AHR is overexpressed and chronically activated in certain types of cancer — including glioblastoma, breast cancer, oral, and gastric cancers. Chronic AHR activation can suppress tumor-specific immunity and trigger stem-like cancer cell formation, driving tumor
migration, proliferation, and metastasis. Cancer cells express high levels of indoleamine 2,3-dioxygenase (IDO1) and tryptophan 2,3-dioxygenase (TDO2), generating tryptophan-derived AHR agonists that suppress protective anti-tumor immune responses (e.g., glioblastoma cells activate AHR in tumor-associated macrophages).
Along with the clinical trials with tapinarof, Table 2 [see attached review] lists more than 20 other clinical trials that have either been completed, or are underway, to interrogate the clinical effects of AHR modulation — using endogenous and dietary ligands as well as synthetic small molecules. Fig. 3 is a diagram illustrating how probiotics might target AHR and related pathways. Fig. 4 demonstrates how AHR-targeting nanoparticles might be used in immunomodulation. Table 3 shows drugs that are “currently under preclinical development”; this includes three chemical structures (two of which are focused on solid tumors), plus three (chemical structures not available) that are focused on inflammatory and autoimmune diseases, viral infections and cancer, and inflammatory bowel disease, respectively.
In conclusion, authors state that “historically considered a liability target due to its initial identification as a receptor for man-made environmental toxicants, AHR has now emerged as a viable therapeutic target for inflammatory, infectious, cardiovascular, and neoplastic diseases.” Development, approval, and commercialization of tapinarof has validated AHR as a drug target, and additional AHR-targeting medicines are expected to be developed soon. Once avoided as a liability target, AHR has now emerged as a druggable target for therapeutic immunomodulation. Identification of important roles for AHR in the physiological regulation of the immune response has encouraged and guided the development of AHR-targeting drugs. The commercialization of tapinarof has provided validation to the biopharmaceutical industry to pursue additional drugs focused on modulating AHR biology. An improved understanding of structure–activity relationships should lead to more efficient development of safe and efficacious AHR-targeted immunotherapies.
Sorry that this email blog is so long — but this review is among the most comprehensive, detailed and information-rich summaries that GEITP has ever encountered (and this includes reviews on any topic).
DwN