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. 2021 Apr 6;12(6):902–914. doi: 10.1039/d1md00015b

Recent advances in the development of AHR antagonists in immuno-oncology

Lijun Sun 1
PMCID: PMC8221258  PMID: 34223158

Abstract

The arylhydrocarbon receptor (AHR) is a ligand activated transcription factor that controls the expression of a number of immunosuppressive signaling molecules, including the immune checkpoint proteins PD-1/L1 and cytokine IL-10. AHR activation also stimulates the formation and recruitment of tolerogenic dendritic cells, tumor associated macrophages, and regulatory T cells in the tumor microenvironment, which restrains antitumoral immune response. Overexpression of AHR has been observed in a number of different types of cancer and suggested to contribute to immune dysfunction and cancer progression. One prominent endogenous ligand of AHR is the oncometabolite kynurenine, a product of tryptophan metabolism catalyzed by the dioxygenases IDO1 and TDO that are often aberrantly activated in cancer. AHR has gained significant interest as a drug target for the development of novel small molecule cancer immunotherapies, as evidenced by the advancement of two clinical candidates into phase 1 clinical trials in patients with advanced cancer. Discussed in this Review is a brief background of AHR in immuno-oncology and the recent progress in the discovery and development of AHR antagonists.

This review discusses the rational and recent progress in targeting the transcription factor AHR for the discovery and development of novel small molecule immunotherapies for the treatment of cancer.graphic file with name d1md00015b-ga.jpg

1. Introduction

Contemporary approaches to the discovery and development of anticancer drugs can be classified into two major categories: molecularly targeted therapies and immunotherapies. Targeted therapies are rationally designed based on a specific gene mutation intrinsic to cancer cells, and the best examples include tyrosine kinase inhibitors (TKIs) of the EGFR signaling pathway.1 Acquired resistance to TKI emerges quickly and frequently, due to treatment induced secondary mutations, which might be overcome by next generation therapeutics that inhibit the enzymatic activity of the mutated kinase.2 Cancer immunotherapies do not directly act on cancer cells, but instead exploit the immune system to eradicate cancer and control tumor growth.3 The regulatory approval of a number of immune checkpoint inhibitors, monoclonal antibodies that target the cell surface receptors programmed death receptor/ligand 1 (PD-1/PD-L1) or the cytotoxic T lymphocyte associated protein 4 (CTLA-4), illustrates the success in the treatment of late stage cancer by activating T cell mediated adaptive immunity. Cancer cells can induce the expression of CTLA-4 or PD-1 in T cells to negatively regulate their anticancer effector functions. By disrupting the tumor supportive signals imposed by cancer cells, immune checkpoint inhibitors enable the body to mount durable antitumor immunity that leads to long-lasting disease remission in advanced cancer patients. Despite the unprecedented success in a small portion of cancer patients, a significant majority of patients does not respond to immune checkpoint inhibitors.4 And recent studies have demonstrated that numerous cancer intrinsic and extrinsic mechanisms and signaling pathways within the tumor microenvironment (TME) are implicated in aiding cancer cells to evade immune recognition, which have led to a number of novel targets for the development of cancer immunotherapies against a repertoire of immune checkpoints proteins and beyond.5 Our accumulating knowledge convincingly supports the notion that tumor-infiltrating myeloid and lymphoid cells contribute to the cancer supportive dysfunctional immunity, which exhibit an immunosuppressive phenotype that fails to recognize and induce apoptosis of cancer cells.6–9 Therefore, novel strategies aimed to reverse the TME from immunosuppressive and protumoral to pro-inflammatory and antitumoral phenotypes have been explored for developing cancer immunotherapy for patients that are refractory to existing therapies. This Review attempts to summarize the recent progress in the development of small molecule anticancer immunotherapies by targeting the arylhydrocarbon receptor (AHR) signaling pathway. Comprehensive discussions on other target classes attainable by small molecules can be found in a number of recently published review articles.10–12

2. The role of AHR in modulating immune responses in cancer

AHR is an evolutionally conserved, ligand activated transcription factor that regulates the expression of a number of genes involved in the controls of immune responses in cancer (Fig. 1). AHR is expressed in epithelial cells and specific subsets of immune cells, and the functions as well as the interactions among these cells are shaped by AHR signaling. AHR is a member of the basic helix–loop–helix (bHLH) Per–Arnt–Sim (PAS) superfamily and contains a ligand-binding domain in its PAS-B region. In the absence of binding to a small molecule ligand, inactive AHR is sequestered in the cytosol by the chaperon heat shock protein-90 (HSP-90) and AHR-interacting protein (AIP), among others. Upon activation by ligation with agonists, AHR undergoes conformational changes, translocates to nucleus and dimerizes with the AHR nuclear translocator (ARNT) to initiate what is defined as its canonical genomic signaling pathway. The AHR–ARNT complex binds to a xenobiotic-response element (XRE) of the promoter regions of its corresponding target genes to regulate their expression. Notably, the genes of immune checkpoint receptors PD-1 and PD-L1, which are drug targets for a number of successful cancer immunotherapies including nivolumab, pembrolizumab, and durvalumab, contain multiple XRE elements and their expressions are driven by AHR activation.13,14

Fig. 1. The genomic signaling pathway of AHR. AHR agonists encompass tryptophan metabolites, diet-derived organic compounds, synthetic chemicals and pharmaceutical agents. AHR antagonists compete for binding to AHR to prevent its activation.

Fig. 1

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The tumor tissue is composed of proliferating cancer cells and diverse immune cells that constitute the tumor immune microenvironment (TIME), including the antigen presenting dendritic cells (DCs), tumor associated macrophages (TAMs), myeloid derived suppressor cells (MDSCs), and T lymphocytes. Cancer cells develop multiple mechanisms to suppress tumor-specific immune responses, such as the recruitment and induction of TAMs, MDSCs, and regulatory T (Treg) cells in the TME, as well as the expression of immune checkpoints and immunosuppressive molecules. AHR is expressed in both cancer cells and tumor infiltrating immune cells and directly impacts their responses to cancer development and progression15 (Fig. 2). There is strong evidence indicating that AHR activation in immune cells by exogenous or endogenous agonists confers immunosuppression. For instance, AHR expression is significantly higher in human tumor biopsies that demonstrate a tumor growth factor-β (TGF-β) dominant immune signature, which predicts the poorest prognosis and is associated with high TAMs in the TME.16 Highlighted in the next sections are some of the significant findings connecting AHR activation to impairment of antitumoral innate and adaptive immune responses (reviewed in ref. 17–19). The investigation of AHR in immunity has been facilitated by a number of AHR ligands (Fig. 3), often used as in vitro and in vivo pharmacological tools. A detailed discussion of the structural diversity, ligand and cell contexture dependent functional relationship of AHR ligands can be found in a review article referenced here.20

Fig. 2. Multiple mechanisms are involved in the contributions of AHR to the recruitment and induction of tumor promoting immune cells from both the myeloid and lymphoid lineages.

Fig. 2

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Fig. 3. Chemical structures of representative tool compounds commonly used in studying the biological function of AHR. ITE: 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester; FICZ: 6-formylindolo[3,2-b]carbazole; TCDD: 2,3,7,8-tetrachlorodibenzodioxin; BaP: benzo(a)pyrene; VAF247: (4-(3-chlorophenyl)-pyrimidin-2-yl)-(4-trifluoromethylphenyl)-amine; CH223191: 1-methyl-N-[2-methyl-4-[2-(2-methylphenyl)diazenyl]phenyl-1H-pyrazole-5-carboxamide; ANF: alpha-naphthoflavone.

Fig. 3

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2.1. AHR as a key effector molecule of tryptophan metabolism

Significant insights in our understanding of the role of AHR in immuno-oncology have been obtained from investigating the functions of indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO), which are frequently overexpressed in cancer and induce immune suppression by depletion of tryptophan and the generation of tryptophan metabolites as signaling molecules (reviewed in ref. 21 and 22). IDO1 and TDO are the rate-limiting enzymes in the kynurenine (KYN) pathway that metabolizes the essential amino acid tryptophan to produce KYN, a bona fide endogenous AHR agonist (Fig. 4). IDO1, on the other hand, is a target gene of AHR and is upregulated by KYN mediated AHR activation. Therefore, aberrant tryptophan metabolism in cancer amplifies an IDO1–KYN–AHR metabolic-signaling circuitry that sustains an immunosuppressive and tumor supportive TME (reviewed in ref. 23 and 24). The heightened tryptophan metabolism because of overexpression of IDO1 and/or TDO in cancer cells produces high concentration of KYN. Cancer cell derived KYN activates AHR not only in cancer cells in an autocrine manner, but also in a paracrine manner in neighboring immune cells (as detailed in section 2.2).25–27 In immune competent mice bearing B16 melanoma that expressed high levels of IDO1/TDO, AHR upregulation in DCs, TAMs and Tregs was associated with their immunosuppressive phenotypes. Treatment of mice bearing B16 tumor with overexpression of either IDO1 or TDO with the AHR antagonist CH223191 (Fig. 3) inhibited tumor growth. CH223191 was however ineffective in mice bearing the wild-type B16 melanoma, likely due to low levels of AHR signaling.16 Therefore, it is interesting from a viewpoint of ‘targeted cancer immunotherapy’ that the expression of IDO1/TDO might be evaluated as biomarkers to assist melanoma patient stratification for testing the therapeutic potentials of AHR antagonists.

Fig. 4. Endogenous AHR agonists generated from tryptophan (Trp) catabolism. (A) The kynurenine pathway is initiated by IDO1/TDO to produce kynurenine (KYN) via oxidative ring opening of the pyrrole moiety. KYN is a common intermediate to kynurenic acid (KYNA) (catalyzed by KAT) and 3-hydroxyanthranilic acid (3-HAA) (catalyzed by KMO and then KYNU). KAT: kynurenine aminotransferase; KMO: kynurenine-3-monooxygenase; KYNase: kynureninase. (B) The indole-3-pyruvate pathway is initiated by IL4I1 (IL-4 induce gene 1) via an oxidative deamination reaction to produce indole-3-pyruvate acid, which is further metabolized to form KYNA and indole-3-aldehyde.

Fig. 4

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In addition to KYN, tryptophan catabolism generates additional signaling molecules that directly or indirectly act on AHR (Fig. 4). Kynurenic acid (KYNA), a downstream metabolite of KYN catalyzed by kynurenine aminotransferases (KAT), is a more active AHR agonist than KYN (KD: 1.6 and 4 μM for KYNA and KYN, respectively).17,28–30 Furthermore, KYNA is also produced in the indole-3-pyruvic acid pathway of tryptophan metabolism catalyzed by the amino acid oxidase IL-4 induce gene 1 (IL4I1). Further, indole-3-pyruvic acid is a source of the AHR agonist indole-3-aldehyde, which can also be produced by gut microbial degradation of tryptophan. Upregulation of IL4I1 in the brain tumor glioblastoma (GBM), as well as in other types of cancers such as chronic lymphocytic leukemia (CLL), increased the levels of KYNA, which suppressed T cell proliferation that could be reversed by AHR inhibition.31 KYN metabolism also produces the immunosuppressant 3-hydroxyanthranilic acid (3-HAA), catalyzed by kynureninase (KYNU) and kynurenine 3-monooxygenase (KMO) (Fig. 4). 3-HAA is not a direct AHR agonist but instead dose-dependently enhanced AHR activation by KYN to potentiate the induction of Treg cells.32 In summary, AHR is a critical effector molecule to link dysfunctional immune responses to aberrant tryptophan metabolism in the TME.

2.2. Regulation of immune checkpoint proteins by AHR

Costimulatory signals are required for T cells to mount an effective antitumoral response, which is adversely disrupted by the expression of negative immune checkpoint ligands and their receptors including PD-1/L1. Cigarette smoking is a major cause of non-small cell lung cancer (NSCLC) cells. BaP (Fig. 3), an AHR agonist found in cigarette smoke and fossil fuel exhaust, dose-dependently induced the expression of PD-L1 (B7-H1) in NSCLC cells, and this effect was AHR-dependent and inhibited by the AHR antagonists CH223191 and ANF (Fig. 3). In a syngeneic murine lung cancer model, treatment of C57BL mice inoculated with Lewis lung carcinoma (LLC) with ANF decreased PD-L1 expression and inhibited tumor growth, which was accompanied by increased number of CD8+ T cells and higher levels of the cytotoxic cytokine interferon-γ (IFN-γ) in the lungs. Further, ANF significantly enhanced the anticancer activity of an anti-PD-L1 antibody in the murine lung (LLC) and colon (MC38) cancer models that are sensitive to immune checkpoint inhibitors, as well in the fibrosarcoma (Ag104Ld) model that is refractory to anti-PD-L1 antibody monotherapy. Importantly, NSCLC patients expressing higher levels of AHR achieved better disease control than those with low levels of AHR when treated with pembrolizumab,13 suggesting AHR as a possible biomarker to select patients who might be more likely to respond to immune checkpoint inhibitors.

Cancer stem-like cells (CSCs) are a subpopulation of cancer cells characterized by resistance to anticancer therapies and their propensity to metastasize and repopulate tumors with proliferating cancer cells. CSCs were shown to selectively upregulate IDO1 and maintain a high concentration of KYN.33 Further, KYN derived from CSCs was transported to tumor infiltrating cytotoxic T lymphocytes (CTLs) that express AHR. AHR activation by KYN upregulated the immune checkpoint protein PD-1 in CTLs, which was suppressed by the AHR antagonist CH223191.14 Similarly, cancer cells from the brain tumor GBM were also shown to generate excess amount of KYN by upregulating TDO, and to suppress effector functions of immune cells via AHR activation by KYN.29 Specifically, the KYN-AHR signaling axis upregulated the expression of CD39 in GBM infiltrating TAMs to impair the cytotoxicity of CTLs.34 CD39 is ectonucleotidase that catalyzes the hydrolysis of extracellular ATP to form the potent immunosuppressant. It is worth noting that CD39, an immune checkpoint molecule, is under intensive investigation as a novel target for the development of cancer immunotherapies, adenosine.35 Moreover, activation of AHR in DCs was also shown to induce immunosuppressive functions in part by significant upregulation of B7-H4, a negative costimulatory molecule expressed by cancer cells and antigen presenting cells and an emerging target subjected to therapeutic intervention in immuno-oncology.36,37 In summary, AHR contributes to the overexpression of negative costimulatory molecules that suppress anticancer cellular immunity.

2.3. Regulation of IL-10 and regulatory T cells by AHR

The immunosuppressive regulatory T (Treg) cells play an important role in the containment of inflammatory responses during infection to avoid autoimmunity that might cause tissue damage. Cancer cells can hijack this self tolerant process to suppress antitumoral immune responses. The presence of Treg cells in tumor tissue confers to resistance to immune checkpoint inhibitors and poor survival. AHR plays an important role in the development of Treg cells as it is expressed in type 1 regulatory T (Tr1) cells and is suggested to regulate the expression of the transcription factor foxp3 in FOXP3+ Treg cells.38 For example, the endogenous AHR agonist KYN was shown to promote the development of Tregs through the activation of AHR.30 AHR activation in antigen presenting DCs, which processes cancer derived neoantigens to initiate cancer specific programs in adaptive immunity, by the AHR agonists TCDD or VAF347 (Fig. 3) led to a tolerogenic phenotype that is ineffective in the induction of anticancer immunity, characterized by upregulation of the immunosuppressive cytokine IL-10 and tryptophan catabolic enzyme IDO1.39–42 IL-10 is a potent immunosuppressive mediator that promotes the development of Treg cells, which expresses AHR and supports tumor growth in part by impairing the maturation and the antitumoral effector function of CTLs. AHR activation in Treg cells by TCDD or other AHR agonists drives IL-10 production to limit inflammation.43,44 Furthermore, IL-10 promotes the development and proliferation of tolerogenic DCs, TAM, and MDSCs in the TME to support angiogenesis and immune escape of cancer cells.45–47 In summary, AHR is a key transcriptional regulator that induces the expression of the anti-inflammatory cytokine IL-10 by DCs and Treg cells, which helps to shape a tumor supportive TME.

2.4. Regulation of NK cell effector function by AHR

Natural killer (NK) cells are presented in the TME and constitute an important component in cancer immune surveillance48 Although less is known regarding the role of AHR in regulating the effector function of NK cells, AHR is expressed in NK cells and seems to display diverse activity that is cell contexture dependent. AHR deficient NK cells exhibited diminished capacity to control RMA-S (murine lymphoma) tumor formation in vivo, while mice treated with the AHR agonist FICZ improved control of tumor formation49 Conversely, human acute myeloid leukemia (AML) cells were shown to generate AHR agonists that in a paracrine manner activated AHR in NK cells to impair their anticancer activity, which could be restored by the AHR antagonist CH223191.50 Considering the importance of NK cells in cancer development, better understandings of the role of AHR in NK cells will help develop more effective strategies for targeting NK cells in immuno-oncology.

2.5. Regulation of chemokines and chemokine receptors by AHR

Recruitment of immune cells to the TME is controlled by chemokine-chemokine receptor mediated signaling for cell trafficking. In breast cancer harboring the BRCA1 mutation, AHR signaling was involved in attracting TAM into the breast cancer TME and there was a positive correlation between the expression of AHR and that of the chemokines CXCL1/2 and CCL2/5 in human breast cancer tissue.51 AHR activation by TCDD in macrophages induced the chemokine monocyte chemoattractant protein-1 (CCL2) and IL-8 (CXCL8), ligands for the chemokine receptor CXCR2 expressed by monocytes and neutrophils to guide their migration.52 In GBM, AHR in cancer cells and tumor infiltrating macrophages appeared to coordinate the recruitment of monocyte derived macrophages to the TME via upregulation of CCL2 and CCR2, respectively.34 Mice exposed to TCDD produced large amount of peritoneal MDSCs that exhibited AHR-dependent upregulation of CCR2, CCR5, and CXCR2. The TCDD induced MDSCs displayed potent immunosuppressive activity, inhibited T cell proliferation in vitro, and increased IL-10 and TGF-β in adoptive MDSC cell transfer mouse models.53 Moreover, AHR controls the expression of the chemokine receptor GPR15 in Treg cells to regulate their recruitment to the large intestine during inflammation.54

In conclusion, large bodies of literature reports indicate that AHR is a molecular sensor that detects aberrant tryptophan metabolism and toxic chemicals. The activation of AHR by both endogenous (KYN) and exogenous (BaP) ligands enhances immunosuppressive signaling pathways (e.g. PD-1, IDO1, and IL-10) that impair the anticancer effector function of tumor-infiltrating innate and adaptive immune cells. AHR functions as a communication hub to coordinate the multilateral signals between cancer cells and tumor-infiltrating immune cells. Furthermore, AHR signals the recruitment of immune cells to tumor tissues by regulating the expression and chemokines and their receptors. Therefore, inhibition of AHR signaling can potentially reverse the cancer supportive TME and represents a promising approach for the development of novel small molecule anticancer immunotherapies.

3. AHR antagonists – from tool molecules to therapeutic candidates

A number of in vitro assays have been established to characterize compounds that inhibit AHR signaling. The cell based XRE reporter luciferase assay is readily scalable for high-throughput (HTS) screening and routinely used to determine the IC50 of AHR antagonists in inhibition of ligand induced AHR activation. AHR activation upregulates the gene expression of the cytochrome P450 1A1 and 1B1 (CYP1A1 and CYP1B1), and the measurement of their mRNA levels by qPCR has also been widely used as a cellular readout for AHR singling. In these cell based assays, some AHR antagonists were reported to decrease AHR signaling below baseline levels in the absence of an exogenous agonist, which could be indicative of a mechanism of inverse agonism or simply due to the presence in assay medium of endogenously generated AHR agonists (e.g. tryptophan metabolites). Tritium labeled TCDD (3H-TCDD) and a radioactive AHR photo affinity ligand (PAL, Fig. 5) have been characterized and available for competitive binding assay with AHR proteins obtained from cell lysate.55 Finally, measurement of AHR nuclear vs. cytosolic localization, as well as formation of AHR-ARNT complex, has been performed as confirmatory assays to corroborate on-target activities by a testing compound.

Fig. 5. The photo affinity ligand (PAL) of AHR. 2-Azido-3-[125]iodo-7,8-dibromodibenzo-p-dioxin.

Fig. 5

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Structurally distinctive AHR antagonists with improved pharmacological properties and pharmacokinetics have been discovered through structural modification of tool compounds (e.g. CH223191), HTS and ligand based lead optimization, resulting in the selection of development candidates for preclinical and clinical investigations (Table 1). In the next sections, AHR antagonists disclosed in literature reports and patent applications will be categorized and discussed according to their structural similarity, with a focus on representative examples that have been described with a more comprehensive data set. Moreover, the structural biology of AHR is summarized with an attempt to stimulate computer aided drug designs.

AHR antagonists under preclinical and clinical development.

Compound code IC50 (nM) Development status ClinicalTrials.gov identifier Sponsor
SR-1 127 Phase 1/2 (results reported) NCT02765997 Novartis
BAY2416964 22 Phase 1 (enrolling patients) NCT04069026 Bayer
IK-175 — Phase 1 (enrolling patients) NCT04200963 Ikena Oncology
BAY-128 39.9 Preclinical — Bayer
PX-A24590 60 Preclinical — Phenex
KYN-101 22 Preclinical — Ikena Oncology
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3.1. AHR antagonists sharing a purine core and related scaffolds

The purine derivative SR-1 (Fig. 6) is a selective human AHR (hAHR) antagonist and the first in its class to have been evaluated in humans as an ex vivo agent to expend blood cells for transfusion. It potently inhibited TCDD mediated hAHR activation (IC50: 127 nM) and PAL binding to hAHR protein (Ki: 40 nM).56 SR-1, via inhibition of AHR signaling, dose-dependently potentiated the expansion of human hematopoietic stem cells (HSCs) co-stimulated by a cocktail of cytokines in vitro. Transfusion of HSCs is a promising treatment option for cancer patients with hematological malignancy, and a phase I/II clinical trial was conducted to evaluate the efficacy of transfusion of HSCs promoted by SR-1 ex vivo (NCT02765997). Preliminary report indicated that addition of SR-1 to a cytokine cocktail in cell culture led to a 330-fold increase in HSCs, which were successfully engrafted in cancer recipients.57

Fig. 6. AHR antagonists with a purine core and related scaffolds. (+) and (−) 1 are examples 65a and 65b in WO2020081636, respectively; 2 is example 24 in WO2018191476; 3 is example 8 in WO2020050409); 4 is example 12 in WO2020039093.

Fig. 6

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GNF-351 (Fig. 6), a close structural analogue of SR-1, displayed similar activity to SR-1 in inhibition of PAL binding to hAHR protein (IC50: 62 nM vs. 80 nM for SR-1 in the same assay). It inhibited AHR activation by TCDD more potently in human than in mouse hepatocytes, with an IC50 of 8.5 and 116 nM, respectively.58 GNF-351 was however not orally bioavailable in mice and failed to prevent AHR activation in mice after oral administration. The lack of oral exposure is likely due to poor permeability, as well as rapid P450 mediated oxidative metabolism of the indole moiety of GNF-351.59

The pyrazolo[1,5-a]pyrimidine analogue KYN-101 (Fig. 6) was reported as a potent AHR antagonist (IC50: 22 nM) and exhibited tumor growth inhibition when dosed orally in syngeneic mouse models of B16 melanoma over expression IDO1/TDO.16 KYN-101 is among a number of novel substituted 5,6-fused bicyclic heteroaromatics described in the patent WO2018195397 filed by Ikena Oncology. IK-175 (structure not disclosed) is an AHR antagonist that shares a similar scaffold to KYN-101. Ikena has initiated a phase 1 clinical trial to evaluate the safety and pharmacokinetics of IK-175 as an immnotherapy in patients with advanced cancer (NCT04200963). Notably, a class of tetrahydrocarbazole substituted pyrazolotriazines was disclosed in patent applications, as exemplified by the chiral molecule 1 (WO 2020081636) (Fig. 5). Both (−) and (+) diasteromers were claimed to inhibit agonist induced activation of AHR in XRE reporter assays, however the exact IC50 was not described.

A class of imidazo[1,2-a]pyrazine derivatives were reported as AHR antagonists by Magenta Therapeutics, as exemplified by the chiral compound 2 (Fig. 6) (WO2018191476). With an IC50 of 11 nM, compound 2 as a racemic mixture is 17.5 times more potent than SR-1 (IC50: 193 nM in the same assay) in inhibiting AHR activation by the AHR agonist VAF347 (Fig. 3). Further, there is a 2-fold separation between the IC50 of the two enantiomers (6 nM vs. 12 nM), indicative of a modest effect of stereochemistry of this class of antagonists on AHR inhibition. Further improvement in inhibitory activity of AHR activation was achieved in a class of compounds containing a tricyclic fused purine core (WO2020050409), including compound 3 (Fig. 6, IC50: 0.11 nM) with an ortho-fluorophenyl group on the phenol moiety. Lastly, AHR inhibition is not limited to compounds with the 5,6-fused aromatic cores. The high potency of the 6,6-fused pyrimidinopiperidine derivative 4 (Fig. 6, IC50: 1 nM) suggests that more drastic structural modifications of the fused bicyclic core are attainable for highly active AHR antagonists (WO2020039093).

3.2. 1,3-Diaryl-pyrazin-6-one-5-carboxamides

The pharmaceutical company Bayer AG has recently disclosed in a series of conference proceedings a class of 1,3-diaryl-pyrazin-6-one-5-carboxylic amides as novel AHR antagonists60–62 (Fig. 7). This novel scaffold was identified via an HTS campaign that involved in the screening of 4 million compounds in cell-based assays. Lead optimization, with emphasis on ligand efficiency and lipophilic property, led to the selection of the development candidate BAY2416964 and the preclinical lead BAY-218 (Fig. 7). Patients with advanced cancer are being enrolled in a phase 1 clinical trial to determine the safety and pharmacokinetics of BAY2416964 (NCT04069026). In preclinical studies, the compound is orally bioavailable (F: 83% in mouse) and efficacious in inhibition of tumor growth in syngeneic mouse models of colon cancer (CT26) and melanoma (B16). BAY2416964 exhibits similar potency in inhibition of AHR activation in human (IC50: 22 nM) and mouse (IC50: 15 nM) cellular assays. The chiral center of the amide side chain in this class of molecules has shown a more profound impact on its biological activity. For example, the difference in AHR inhibitory activity between the (+) and (−) enantiomers of compound 5 (WO2018146010) is >85-fold (IC50: 1.5 and 130 nM, respectively). Similarly, BAY-218 is 75-fold more potent (IC50: 39.9 nM) than its (R-) enantiomeric isomer (IC50: 302 nM). In preclinical studies, BAY-218 was shown to stimulate antitumoral immune responses as evidenced by increases of CTLs and natural killer (NK) cells and decreases of MDSCs and TAMs in tumor tissues, which collectively contributed to tumor growth inhibition of BAY-218 as a monotherapy, was well as enhancement of efficacy of an anti-PD-L1 antibody as a part of combination therapy.

Fig. 7. AHR antagonists of the 1,3-diaryl-pyrazin-6-one-5-carboxamide chemotype. BAY2416964 is example 17 in WO2018146010; BAY-218 and its R- enantiomer are examples 23 and 24 in WO2017202816, respectively; the (−) and (+) enantiomers of 5 are examples 34 and 35 in WO2018146010, respectively.

Fig. 7

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3.3. Pyrazole-5-carboxylic arylamides

As discussed above, the AHR antagonist CH223191 (Fig. 3) is a widely used tool compound to study AHR pharmacology. With an IC50 of 1.15 μM in inhibition of 3H-TCDD binding to AHR protein, CH223191 is moderately active but provides a useful template for structural modifications to discover more potent AHR antagonists.63 Modifications have been carried out with an aim at replacing the diazo linker group, which is labile to metabolic fragmentation mediated by commensal bacteria in the gastrointestinal track. Thus, a ring fusion strategy to the middle benzene ring led to a class of indole derivatives as AHR antagonists, including the preclinical candidate PX-A24590 (Fig. 8) developed by Phenex Pharmaceuticals (WO2018141857). PX-A24590 is an orally efficacious AHR antagonist (IC50: 60 nM). In syngeneic mouse models of pancreatic (Panc02) and colon (CT26 and M38) cancers, daily oral administration of PX-A24590 demonstrated tumor growth control as a single agent, and enhanced the anticancer activity of an anti-PD-L1 antibody in colon cancer or the chemotherapy gemcitabine in pancreatic cancer. Further, tumor infiltrating CTLs were increased in pancreatic tumor treated with PX-A24590 as compared to vehicle control.64 By an alternative ring fusion to the left side benzene moiety to eliminate the diazo functionality, a class of quinolone analogues was disclosed in WO2020018848 as AHR antagonists including compound 6 (Fig. 8).

Fig. 8. AHR antagonists designed from CH223191. PX-A24590 is example 1 in WO2018141857; 6 is example 52 in WO2020018848.

Fig. 8

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3.4. Chalcone and stilbene derivatives

The chalcone derivative 7 (Fig. 9) was identified as a potent AHR antagonist by modifying an HTS hit discovered by screening a 6400-member compound library in a zebra fish CYP1A1 assay.65 In an in vitro luciferase reporter assay using COS-7 cell line, 7 (IC50: 8.3 nM) inhibited AHR activation more potently than CH223191 (IC50: 98 nM).

Fig. 9. Chalcone type AHR antagonists.

Fig. 9

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The polyphenol natural product resveratrol 8a (Fig. 10) is a mixed ligand of AHR, which inhibits AHR activation at low concentration but activates AHR at higher concentrations.66 SAR studies focusing on the modification of the OH groups in the stilbene scaffold resulted in a highly potent AHR antagonist 8b (IC50: 1.4 nM), as compared to an IC50 of 169 nM for resveratrol under the same assay conditions.67 Strikingly, substitution of the 4′-chloro in 8b (R1: Cl) with a trifluoromethyl group in 8c (R1: CF3) led to reversal of AHR receptor inhibition by 8b to AHR activation by 8c (EC50: 0.4 nM).

Fig. 10. Stilbene type AHR ligands.

Fig. 10

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3.5. Thiazolidinones

A similar reversal of mode of action, i.e. inhibition vs. activation of AHR signaling by structurally similar ligands, was observed in a class of thiazolidinone analogues (Fig. 11).68 For example, compound 9a, which did not exhibit AHR activation at doses as high as 100 μM, inhibited TCDD induced expression of CYP1A1 in mouse hepatocytes more potently than CH223191 (IC50: 1.5 and 7.1 μM for 9a and CH223191, respectively). Conversely, the analogue 9b induced AHR activation with an EC50 of 6.6 μM.

Fig. 11. Thiazolidinone type AHR ligands.

Fig. 11

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3.6. Flavonoids

Together with the aforementioned ANF (Fig. 3), natural and synthetic flavonoids represent another class of AHR antagonists, including 3′-methoxy-4′-nitroflavone (MNF; IC50: 2.27 nM) (Fig. 12), 5,7,-dimethoxyflavone (DMF; IC50: 0.8 μM), and 6,2′,4′-trimethoxyflavone (TMF; IC50: 1 μM).69–72 The synthetic flavone CB7993113 (Fig. 12) was identified as an AHR antagonist (IC50: 0.33 μM) via docking to a homology model of the AHR ligand binding domain.73 It competed with TCDD for direct binding to both human and murine AHR, and at 10 μM concentration completely blocked agonist-induced AHR nuclear translocation. Preliminary PK study indicated that it was orally available with a plasma half-life of 4 h in mice. Moreover, AHR inhibition by CB7993113 or CH223191 enhanced the cytolytic potency of NK cells by promoting expression of CD94, TRAIL, perforin and granzyme B. Pretreatment of multiple myeloma cells (MM) with the AHR antagonists increased their susceptibility to NK cell cytotoxicity and NK cell mediated antibody-dependent cell-mediated cytotoxicity (ADCC) by daratumumab (anti-CD38 antibody) and elotuzumab (anti-CD319 antibody), which are approved by FDA for the treatment of MM.74 It was shown that CB7993113 significantly inhibited tumor growth and extended survival of mice bearing orthotopic oral squamous cell carcinoma.75

Fig. 12. Flavonoids as AHR antagonists.

Fig. 12

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3.7. Reposition of existing pharmaceutical agents

Mechanistic profiling investigations have identified AHR antagonists among FDA approved drugs and clinical candidates, providing opportunities to repurpose them for cancer immunotherapy. Clofazimine (Fig. 13), an FDA approved drug for the treatment of leprosy, was shown to inhibit AHR activation by TCDD and BaP at low micromolar concentrations in vitro and inhibited tumor growth in mouse models of multiple myeloma and melanoma via oral gavage.76 The kinase inhibitor vemurafenib (Fig. 13) was reported to compete with 3H-TCDD for AHR binding, and inhibit the AHR-dependent activity of TCDD, BaP, and FICZ.77 Interestingly, vemurafenib did not seem to inhibit the nuclear translocation of AHR but instead prevent the XRE binding of the AHR–ARNT complex.78 The utrophin modulator ezutromid (Fig. 13), a development candidate for the treatment of Duchenne muscular dystrophy, was revealed by chemical proteomics investigations as a potent AHR antagonist.79 Cells treated with ezutromid showed retention of AHR in the cytosol, prevention of ITE-induced AHR nuclear translocation, and decrease of CYP1A1 expression. The authors utilized a fluorescence quenching assay to quantify the binding of ezutromid to a purified mouse AHR and obtained an apparent dissociation constant (KD) of 50 nM.

Fig. 13. AHR antagonists identified from studies of pharmaceutical agents.

Fig. 13

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3.8. Structural biology of AHR and its interactions with partnering coregulating protein and DNA responsive element

A major challenge in the design of orthosteric AHR antagonists is that we still lack information at an atomic level the receptor–ligand interactions. AHR protein contains multiple domains involved in protein–protein interaction (PPI), protein–DNA interaction, and binding to small molecule ligands (Fig. 14A).80 Currently, there are no literature reports of crystal structures detailing the ligand binding site in the PAS B domain of AHR. MD simulation and homology modeling have been frequently applied to hypothesize receptor–ligand interactions and to corroborate with findings from structure–activity relationship (SAR) studies (Fig. 14B, top).81,82 But little is known regarding the ligand induced conformational changes of AHR, which presumably plays a key role in ligand-induced activation or inhibition of AHR signaling. Importantly, the structural complexes of truncated AHR without the PAS B domain have been resolved, revealing the AHR/ARNT PPI and AHR/ARNT protein–DNA interactions (Fig. 14B, bottom).83,84 Moreover, the structural information of other members of the bHLH–PAS family, including neuronal PAS 1 (NPAS1) and NPAS3 in complex with ARNT,85 has been reported and reviewed in.86 It is therefore tempting to speculate that ligands interrupting the AHR–ARNT or the AHR–DNA interactions could be designed and characterized as ‘allosteric’ AHR antagonists. Indeed, crystallographic and mutagenesis studies have identified hydrophobic residues of the PAS A domain, which are at the interface between AHR and ARNT PPI.87,88 Furthermore, MD simulations may shed light on the conformational changes imparted by orthosteric ligands not only locally in the PAS B domain, but also in the distal PAS A and the bHLH domains responsible for PPI and protein DNA interaction, respectively. In silico modeling of complex quaternary systems such as AHR–ARNT–DNA will provide invaluable insight in the absence of experimental structural information.

Fig. 14. A: Domain structures of AHR. PAS B domain is also involved in binding to HSP90 proteins. Q-Rich region is involved in transcriptional activation (from the Author's research, as detailed in ref. 82). B: Homology model of the AHR ligand binding domain (top), and the crystal structure (PDB ID: 6v0l) of the quaternary complex of AHR (cyan), ARNT (pink), and nucleic acid (grey) (bottom).

Fig. 14

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4. Conclusion and future perspectives

The tumor infiltrating myeloid cells and lymphocytes are shaped by cancer cells to exhibit a tumor supportive phenotype characterized by immunosuppressive effector functions. AHR has emerged as a central node to convey the communication between cancer cells and immune cells. The AHR genomic signaling pathway, which regulates the expression of soluble chemokines and cytokines as well as cell surface immune checkpoint proteins and chemokine receptors, appears to play a fundamental role in recruiting and promoting the protumoral functions of DC, TAM, MDSC, CTL, NK cell, and Treg cell in the TME. Control of AHR activation can potentially redirect the immune cells toward an antitumoral phenotype, therefore representing a novel therapeutic approach in immuno-oncology. As highlighted in this Review, recent research has led to the discovery and characterization of a number of structurally diverse and highly active AHR antagonists that demonstrate encouraging anticancer activities in preclinical tumor models. Two clinical development candidates targeting AHR inhibition have recently entered phase 1 clinical trials, aiming to define their safety and pharmacokinetic/pharmacodynamics (PK/PD) profiles in cancer patients. Of particular importance from those clinical trials will be the identification of potential PD biomarkers for patient stratification because in preclinical settings the function of AHR is strongly dependent on the tissue and cellular contexture. Furthermore, AHR is indispensable in the maintenance of barrier tissue homeostasis and tissue repair. Deficiency in AHR expression or lack of diet-derived natural AHR agonists exacerbates colitis in preclinical models. It remains to be clarified if dose-limiting toxicity will arise in barrier tissues such as skin and GI track in human subjects treated with AHR antagonists. Alternatively, targeted delivery to tumor via novel drug delivery technology such as nanoparticles could be pursued to enrich exposure in TME. Furthermore, the functional role of AHR in tumorigenesis and cancer immunity is incompletely understood and there are contradicting reports confounding its oncological and immunological functions. For example, AHR has been described as a tumor suppressor or an oncogene, depending on the types of cancer and study cohorts in the same type of cancer. AHR signaling, depending on the ligand applied, can either suppress or promote autoimmunity via induction of Treg or TH17 cells.89–91 And the AHR agonist FICZ is efficacious in control of tumor growth in syngeneic mouse models of lymphoma and melanoma.92 Therefore, better understanding of the role of AHR and its interplay with metabolism in the TME and identification of biomarkers for patient stratification will be beneficial to the development of AHR modulators for cancer immunotherapy. Currently, the SAR analyses of AHR antagonists have been conducted largely from cellular functional results, and little information is available with regards to their mode of action at an atomic level due to the lack of high resolution structural systems. Insights from ligand–receptor interaction and ligand-induced receptor conformational changes will be critical for improving future drug designs in this target class. In the absence of experimental details of AHR–ligand interactions and ligand induced protein conformational changes, site-directed mutagenesis and structure-functional relationship studies have provided valuable information that could be utilized for developing more accurate in silico models to guide medicinal chemistry. The existence of structurally disparate AHR antagonists, ranging from rigid and flat poly-fused molecules such as clofazimine to SR-1 with higher degrees of conformational flexibility as outlined in this Review, indicates that it is highly conceivable that additional novel scaffolds will be designed and discovered. Because of the nature of ligand-dependent modulation of AHR signaling, expanding the chemical space of AHR antagonists will not only increase the likelihood of successful drug development but also provide improved experimental probes for better understanding of the biological functions of AHR.

Conflicts of interest

There is no conflict of interest to declare.

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