Targeting Aryl hydrocarbon receptor for next-generation immunotherapies: Selective modulators (SAhRMs) versus rapidly metabolized ligands (RMAhRLs)

Journal Pre-proof Targeting Aryl hydrocarbon receptor for next-generation immunotherapies: Selective modulators (SAhRMs) versus rapidly metabolized ligands (RMAhRLs) Daniela Dolciami, Marco Ballarotto, Marco Gargaro, Luisa Carlota López-Cara, Francesca Fallarino, Antonio Macchiarulo PII:

S0223-5234(19)30994-8

DOI:

https://doi.org/10.1016/j.ejmech.2019.111842

Reference:

EJMECH 111842

To appear in:

European Journal of Medicinal Chemistry

Received Date: 5 September 2019 Revised Date:

30 October 2019

Accepted Date: 30 October 2019

Please cite this article as: D. Dolciami, M. Ballarotto, M. Gargaro, L.C. López-Cara, F. Fallarino, A. Macchiarulo, Targeting Aryl hydrocarbon receptor for next-generation immunotherapies: Selective modulators (SAhRMs) versus rapidly metabolized ligands (RMAhRLs), European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111842. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Masson SAS.

Graphical Abstract

Targeting Aryl Hydrocarbon Receptor for Next-Generation Immunotherapies: Selective Modulators (SAhRMs) versus Rapidly Metabolized Ligands (RMAhRLs).

Authors: Daniela Dolciami,1 Marco Ballarotto,1 Marco Gargaro,2 Luisa Carlota López-Cara,3 Francesca Fallarino,2 Antonio Macchiarulo.1* 1

Department of Pharmaceutical Sciences, University of Perugia, via del Liceo, 1 - 06123, Perugia (Italy).

2

Department of Experimental Medicine, University of Perugia, via Gambuli, 1 – 06132, Perugia (Italy).

3

Department of Pharmaceutical & Organic Chemistry, Faculty of Pharmacy, University of Granada, 18010

Granada (Spain).

Keywords: Aryl Hydrocarbon Receptor; Immunotherapy; RMAhRLs; Selectivity; Specificity; Cancer.

Correspondence author: * Antonio Macchiarulo, PhD. Department of Pharmaceutical Sciences. University of Perugia, via del Liceo 1, 06123 Perugia, Italy. Tel +39 075 585 5160. Fax +39 075 585 5161. e.mail: [email protected]

1

Abstract. Aryl Hydrocarbon Receptor (AhR) constitutes a major network hub of genomic and non-genomic signaling pathways, connecting host’s immune cells to environmental factors. It shapes innate and adaptive immune processes to environmental stimuli with species-, cell- and tissue-type dependent specificity. Although an ever increasing number of studies has thrust AhR into the limelight as attractive target for the development of next-generation immunotherapies, concerns exist on potential safety issues associated with small molecule modulation of the receptor. Selective AhR modulators (SAhRMs) and rapidly metabolized AhR ligands (RMAhRLs) are two classes of receptor agonists that are emerging as interesting lead compounds to bypass AhR-related toxicity in favor of therapeutic effects. In this article, we discuss SAhRMs and RMAhRLs reported in literature, covering concepts underlying their definitions, specific binding modes, structure-activity relationships and AhR-mediated functions.

2

Introduction. Aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor belonging to the basichelix-loop-helix (bHLH) PAS (PER-ARNT-SIM) superfamily of transcription factors[1]. It was discovered and extensively investigated as a ligand-activated transcription factor involved in toxic responses to environmental pollutants such as halogenated aryl hydrocarbons (HAHs; 1-3, Figure 1) and polycyclic aryl hydrocarbons (PAHs, 4-7) [2–9]. The AhR mediated toxicity of these classes of compounds combined with the involvement of the receptor in the regulation of xenobiotic metabolism has contributed to establish reputation among drug hunters of this protein as an antitarget to be avoided in drug discovery programs [10]. Notwithstanding, recent years have witnessed a paradigm shift in this regard, with AhR now experiencing a new lease of life as druggable target associated to novel therapeutic opportunities in immune-related diseases including inflammation and cancer [11–15]. Studies of AhR knockout in mice have indeed provided clues indicating that AhR gene deletion affects several immunological processes such as the development of specific immune cell types, immune tolerance and responses against bacterial infections in mice [16,17]. Moreover, AhR is involved in the differentiation of regulatory T-cells (Treg) and B-cells [18–22]. It has also been reported that the receptor maintains intestinal homeostasis regulating the development of intraepithelial lymphocytes and innate lymphoid cells [17,23,24]. The discovery of endogenous ligands that act as receptor agonists has further corroborated the key role of AhR in normal cell physiology, shifting its functions beyond the original role as merely sensor of environmental chemicals for promoting xenobiotic metabolism. These endogenous compounds include host and microbial diet-derived metabolites, products of indole and tryptophan metabolism [25–27]. For instance, metabolites formed from tryptophan degradation by intestinal microbiota are able to shape immune responses by activating AhR signaling pathways in normal and pathological conditions [28,29]. Although these studies have contributed to unravel the complexity of AhR mediated physiological functions, they also pinpoint that it is a multitude of

3

endogenous ligands produced in different site districts, rather than one single hormone molecule, that accounts for triggering AhR genomic and non-genomic signaling pathways in the organism. Collectively, these findings suggest AhR as the hub of a complex network of genomic and nongenomic signaling pathways that links host’s immune cells to microbiome and environmental factors, shaping innate and adaptive immune processes to environmental stimuli [30,31]. This review article starts with a brief overview of the structure, mechanism of activation and signaling pathways of AhR. Then, we provide the readers with a thorough discussion on two specific classes of AhR ligands: (i) selective AhR modulators (SAhRMs), and (ii) rapidly metabolized AhR ligands (RMAhRLs). These two classes of receptor ligands may indeed yield novel lead compounds for the development of new immunotherapeutic drugs. Receptor binding modes, structure-activity relationships and AhR-mediated functions are highlighted for each class of compounds. Finally, we discuss whether SAhRMs and RMAhRLs may occupy a common region of the chemical space, focusing on similarities and dissimilarities of their structural and physicochemical properties.

4

Figure 1. Prototypical ligands of AhR; chemical stuctures of HAHs (1-3) and PAHs (4-7)

Structure, Activation Mechanism and Signaling Pathways of AhR. As member of the bHLH/PAS superfamily of transcription factors, the sequence of AhR contains a N-terminal bHLH domain that is essential for DNA-interaction (basic region) and dimerization process (HLH) (Figure 2) [32]. Then, the central part of the aminoacidic sequence contains a PAS domain that is composed of two repeated regions of approximately 50 residues, namely PAS-A and PAS-B, with the latter containing the ligand binding pocket [1]. Interestingly, PAS-B domain is also involved in the interaction with Hsp90, with such interaction playing a role in determining ligand binding affinity and specificity [33]. Specifically, this is due to residues of PAS-B domain that, while interacting with Hsp90, are also involved in interactions with receptor ligands [34]. Finally, the C-terminal region of the sequence contains a Q-rich domain, also known as transactivation domain (TAD), that is fundamental for transcriptional activation and interaction with cofactors [35].

Figure 2: Domains of AhR primary sequence.

In general, bHLH/PAS transcription factors are able to regulate the activity of diverse effector domains sensing a large amount of chemical and physical stimuli. Stimulation or repression of transcription is accomplished by hetero-dimerization with conserved bHLH and PAS domains, with differences in C-terminal regulatory regions accounting for different effects [36]. 5

When ligand-unbound, AhR is present in the cytosol as a latent complex composed of two Heatshock proteins 90 (Hsp90), c-Src kinase, p23 and XAP2 chaperones, and AhR-associated protein 9 (ARA9) (Figure 3). Hsp90 proteins are involved both in repression of the intrinsic ability of the receptor to activate DNA transcription and in assisting the ligand binding event [37]. Upon ligand binding-induced release, c-Src kinase triggers fast non-genomic signaling pathways of the receptor. p23 is essential to stabilize the interaction between Hsp90 and AhR [38]. XAP2 (hepatitis B virus X-associated protein) protects the cytosolic form of the receptor against the ubiquitination and provokes a delay in the nuclear translocation of AhR-ARNT complex [39]. ARA9 enhances both the efficiency and potency of AhR agonists [40]. Overall, AhR functions are mediated by genomic and non-genomic signaling pathways. Either pathways start with binding of agonists to the PAS-B domain of the receptor. Triggering genomic signals, cytosolic agonist-bound AhR undergoes conformational changes that lead to exposure of a nuclear localization sequence (NLS) [41,42]. Hence, still associated with cofactors, AhR moves to the nucleus where it displaces from its associated proteins by forming a heterodimeric complex with the Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT). AhR/ARNT heterodimer binds to consensus promoter regions which include AhR Responsive Elements-I (AHREs-I; or Xenobiotic Responsive Elements, XREs; or Dioxin Responsive Elements, DREs; 5′-TNGCGTG-3′) [43] and AhR Responsive Elements-II (AHREs-II, 5’-CATG{N6}C[T|A]TG-3’) [44–46], activating transcription of canonical and/or non-canonical target genes [43,47]. In particular, transcription of canonical target genes is regulated by binding of AhR/ARNT heterodimer to AHREs-I. In liver, AhR canonical target genes include those encoding enzymes of CYP-P450 family (CYP1A1, CYP1A2, CYP1B1), aldehyde dehydrogenase 3 and quinone oxidoreductase, which are collectively associated to xenobiotic metabolism and detoxification [48–50]. In CD4+ immune cells, AhR canonical target genes encode chemokine (4, 5 and 9) and interleukin 12 receptors (β1 and β2), regulating immune system homeostasis [46,48–50]. Non-canonical target genes of AhR can be distinguished in AHRE-II dependent and AHREs independent target genes. Concerning non6

canonical AHRE-II dependent target genes, phylogenetic studies have shown that this group mostly include genes encoding transporters and ion channels [51]. Transcriptional regulation of AHREs independent genes occurs with AhR acting as protein interaction partner of other transcription factors, such as the estrogen receptor (ER), or with AhR/ARNT heterodimer binding to gene enhancer elements that are distinct from AHREs [44,52–54]. Agonist-activated AhR is also involved in the modulation of functions through fast non-genomic signaling pathways by regulating, for instance, intracellular calcium concentration, tyrosine kinase c-Src and MAPK activity [55]. AhR-mediated increase of intracellular calcium concentration contributes to the activation of cytosolic phospholipase A2 (cPLA2), culminating in cyclooxygenase 2 (COX2) activation [56]. In the case of c-Src kinase, this protein is directly associated to AhR cytosolic complex, and is activated upon ligand binding to AhR [57,58]. Activated c-Src is released from AhR complex and accounts for phosphorylation of specific tyrosine residues of protein substrates including the epidermal growth factor (EGF) receptor [59– 61], focal adhesion kinase (FAK), and indoleamine-2,3-dioxygenase 1 (IDO1) [62]. As a consequence, c-Src connects AhR ligand-mediated activation to inflammation, regulation of cell migration and immunotolerance [63–65]. Different mechanisms contribute to switching off AhR-mediated genomic and non-genomic signaling pathways (Figure 3). The dismantlement of the AhR/ARNT heterodimer in the nucleus constitutes a first mechanism that turns off genomic signaling pathways by promoting the shuttling of AhR into the cytosol for proteasomal degradation [66,67]. A second mechanism involves the AhR/ARNT-induced transcription of AHRE-I dependent AHRR gene (Aryl hydrocarbon receptor repressor). AHRR competes with AhR for dimerization with ARNT to form a transcriptionally inactive heterodimeric complex, thereby providing a negative feedback loop of AhR activity regulation.[68] Another negative feedback mechanism relies on the AhR/ARNT-induced transcription of CYP-P450 genes that are involved in the metabolism of xenobiotics. CYP-P450 enzymes reduce cytosolic concentrations of AhR agonists, promoting their degradation by 7

catalyzing oxidative reactions. The combined nuclear and cytosolic actions of AHRR and CYPP450 lead to a fine regulation of the lifetime of AhR transcriptional activity [69,70].

Figure 3: Ligand-induced mechanism of AhR activation for genomic (continuous black arrows) and non-genomic (dashed black arrows) signaling pathways, and switch-off mechanisms of AhR transcriptional activity (continuous red arrows).

AhR Ligands at a Glance Beside HAHs and PAHs (Figure 1), a large array of endogenous and synthetic compounds have been reported in literature to bind and activate AhR [71], including some marketed drugs [72–74]. The wide chemical diversity of these ligands makes outdated the early proposed pharmacophoric model depicting a planar geometry and presence of aromatic/heteroaromatic rings as key features required for AhR agonism (Figure 4) [75]. It rather suggests a binding site receptor with a large volume, diverse interacting residues, extensive hydrophobic contacts and multiple conformations 8

[76–78], that may account for promiscuity of molecular recognition and ligand-specific signalling activities of AhR agonists [79–83].

Figure 4. Early pharmacophore model of AhR ligands based on HAHs and PAHs. Allowed size for length (l = 10-14 Å), medial axis (m < 12 Å) and depth (d < 5 Å) of an ideal AhR ligand are shown according to the study reported by Waller et al [75].

The definition of “agonist” for ligands activating AhR has also been questioned, proposing “modulator” as a more appropiate term on the basis of the following considerations: (i) many of these compounds act as partial agonists and/or full antagonists with cell-specific, tissue-specific and/or species-specific differences [4,84,85]; (ii) some of them have indirect AhR agonistic activity due to inhibition of CYP-P450 enzymes that increases endogenous ligand-mediated AhR activation [86,87]; (iii) others show diverse and specific AhR-mediated signalling activities that makes sketchy their definition as agonist [11,88,89]. A thorough appraisal of these compounds is also complicated by the presence of many endogenous AhR ligands that make difficult the identification and use of a common reference standard for their classification into specific categories of pharmacological activity [90]. Considering these aspects, new emerging pharmacological classes of AhR ligands have been proposed, introducing the definition of Selective AhR Modulators (SAhRMs) and Rapidly 9

Metabolized AhR Ligands (RMAhRLs). Alike some ligands of steroid nuclear receptors [91–94], SAhRMs are those compounds that bind to AhR and activate the receptor for transcriptional regulation of a selected pool of canonical and/or non-canonical target genes, dictating specific biological outcomes [95–98]. This concept also reminds biased ligands for GPCRs, namely compounds able to stabilize receptor/effector complex adopting distinct and specific conformations that preferentially signal through certain pathways relative to others [99–101]. Accordingly, at the molecular level, the interaction of ligands to AhR may promote the stabilization of unique conformations of the transcriptionally active receptor complex that can selectively recruit distinct cofactors and be ultimately associated to differential activation of genomic and non-genomic signaling pathways. Notwithstanding, the lack of crystallographic data for ligand-bound receptor complexes of SAhRMs makes this theory provisional, pending for further validation through more detailed structural works [84]. On the other hand, the definition of Rapidly Metabolized AhR Ligands (RMAhRLs) has been proposed to indicate receptor agonists with specific pharmacokinetic properties of fast metabolic degradation and poor bioaccumulation. The definition grounds on the notion that AhR related toxic effects might be associated to prolonged receptor activation due to poor and/or inefficacious switchoff of its effector signals, rather than transcription of selected target genes mediating toxicity. Indeed, toxic effects were reported in deletion or inhibition studies of CYP1A1, which led to increase metabolic stability of AhR agonists [102,103]. These studies suggest that sustained AhR activation could in part account for adverse effects observed for high metabolic stable and long acting agonists such as halogenated aryl hydrocarbons (HAHs, 1-3) and/or polycyclic aryl hydrocarbons (PAHs, 4-7) [102–104]. Hence, RMAhRLs are ligands that, undergoing rapid inactivating metabolism by AhR-induced CYP-P450s, do not accumulate in the body and have a lower toxicity profile upon acute and/or chronic administration [105]. In this framework, the goal of splitting therapeutic effects from unwanted toxicity in AhR ligands is pursued through the development of SAhRMs and/or RMAhRLs. In the next paragraphs, we report 10

highlights for some examples of these two classes of AhR modulators, focusing on their binding mode, structure-activity relationships and receptor-mediated functions.

Selective AhR Modulators (SAhRMs). Pioneering works of Safe and coworkers were instrumental in the development of first SAhRMs (Figure 5) [106]. They reported two classes of alternate-substituted alkyl polychlorinated dibenzofurans (PCDFs, 8) and substituted diindolylmethanes (DIMs, 9-13) as SAhRMs endowed with antitumor properties for the treatment of breast cancer. Of note, either PCDFs and DIMs were devoid of hepatic CYP1A1-dependent activity or other AhR-mediated latent toxicity, as present in the prototypical potent receptor ligand TCDD (1). On the basis of these finding, authors claimed the development of SAhRMs as viable strategy for the development of novel drugs for the treatment of hormone-dependent cancers, also in combination therapies with tamoxifen or other selective ER modulators [107]. Features pursued in the design of these early SAhRMs were presence of antiestrogenic effects and lack of AhR transcriptional activity at target genes related to TCDD-like toxicity such as CYP1A1 [108]. The most characterized compounds among PCDFs and DIMs are 6-MCDF (6-methyl-1,3,8trichlorodibenzofuran, 8) and 3,3’-DIM (3,3’-diindolylmethane, 9), respectively. They both show partial agonist/antagonist activities at AhR with dissociation constants (Kd) of, respectively, 1.3 x10-10 and 9 x 10-8 M and relative binding affinity (EC50) of 4.9 x 10-8 and 7.8 x 10-5 M, ), compared to that of TCDD (1) [109–115]. Bearing structural elements compliant to the early pharmacophoric model of AhR ligands, 6-MCDF (8) is a synthetic derivative able to displace TCDD (1) from AhR binding pocket. However, at odds with TCDD (1), it forms a nuclear AhR/ARNT complex that does not induce canonical target genes [107]. 6-MCDF (8) was reported to block cancer cell growth and metastasis formation in a transgenic adenocarcinoma of mouse prostate (TRAMP) mice [116,117]. 3,3’-DIM (9) is a natural metabolite of indole-3-carbinole (I3C, 10) that is devoid of planar geometry which was formerly thought as important pharmacophoric feature for AhR agonism. 11

Compound 9 inhibits DMBA-induced mammary tumor growth in Sprage-Delaway rats without inducing hepatic CYP1A1-dependent activity [106]. In addition, 3,3’-DIM (9) was reported to suppress proliferation of human gastric cancer in vitro inducing cell apoptosis and cell cycle arrest [118]. Interestingly, dihalogen-substituted derivatives of 3,3’-DIM (11-13) showed increased antiestrogenic activity being effective at a dose of 1.0 mg/Kg per 2 days in inhibiting growth of DMBA-induced breast cancer cells. Although showing a moderate binding affinity to AhR, these compounds were effective only in AhR-responsive cells, highlighting that antitumorigenic potential was mediated by AhR [119]. These observations support that a planar geometry of ligand molecular shape is not an essential feature for receptor activation. Specifically, as shown in figure 6, compounds 9 and 11-13 show non-planar conformations of energetic minima. Way-166916 (14) is a selective ER modulator (SERM) that showed anti-inflammatory and cardioprotective properties in rheumatoid arthritis and ischemia-reperfusion injury models, respectively [120,121]. Part of these effects were ascribed to its ability in suppressing the transcription of genes of the inflammatory acute phase response (APR). In later studies, Murray and coworkers identified Way-169916 (14) as a dual selective modulator of both ER and AhR. Specifically, they found that suppression of APR genes by the compound occurred in an AhRdependent manner [122]. Compliant to the definition of SAhRMs, Way-169916 (14) proved to lack AhR-mediated transcriptional activity on canonical AHREs-I target genes, including CYP1A1, thereby eliminating risk of latent TCDD-like toxicity. Lead optimization studies around Way169916 (14) were carried out with the aim of keeping SAhRM activity while removing the interaction to ER receptor [80]. Embracing the hypothesis that presence of polar groups are generally detrimental for AhR activity and grounding on the observation that polar groups of 14 make strong hydrogen bonds with ER binding pocket [123], a series of indazole derivatives (15-18) were designed replacing the polar hydroxyl groups of 14 with methoxy moieties at different positions (15-17) and shifting the position of the allyl group between indazole nitrogen atoms (18). As a first result, authors observed an almost total loss of ER binding in compounds 15-18. 12

Moreover, compounds 16 and 17 proved to lack AhR-mediated transcriptional activity on canonical AHREs-I target gene, whereas compounds 15 and 18 still showed AhR agonism activity in the same cellular assay. Competition ligand-binding assay revealed higher affinity of 17 than 16 at AhR receptor, pinpointing most favored positions for activity with substituents at para and meta carbon atoms over the ortho carbon atom of the phenyl ring. Compound 17 was next tested in cellular studies demonstrating its ability to suppress cytokine-mediated inflammatory gene expression, thereby confirming its pharmacological profile of SAhRM with potential anti-inflammatory properties. In vivo proof-of-concept studies confirmed AhR-mediated anti-inflammatory activity of compound 17 in a 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema model [80]. Structural analogues of the AhR partial agonist/competitive antagonist α-naphtoflavone (α-NF, 19) were designed inserting methoxy groups on the B-phenyl ring with the aim of removing stimulation of canonical AHREs-I target genes, while keeping suppression of proinflammatory target genes. This work led to disclose 3′-4’-dimethoxy-α-napthoflavone (DiMNF, 20) which showed the highest activity in competing with TCDD (1) and antagonizing AHREs-I mediated reporter expression, while suppressing proinflammatory genes including SAA1 and members of the complement cascade such as complement factor 3 (C3) [124]. Competitive ligand binding assays revealed similar competition profiles of α-NF (19, IC50 = 25 nM) and DiMNF (20, IC50 = 21 nM) to PAS-B domain of AhR, suggesting that differential agonist/antagonistic effects at AHREs-I mediated gene expression were unlikely to be a consequence of relative binding affinities to PAS-B domain. Of note, docking studies into a homology model of PAS-B AhR pinpointed the formation of an additional hydrogen bond interaction between DiMNF (20), WAY-169916 (14), and Thr283/289 (mouse/human sequences) with respect to α-NF and other prototypical AhR ligands such as TCDD (1) [124]. Such hydrogen bond was proposed to induce a unique conformational change in the receptor leading to a loss of transcriptional activity of AhR complex at canonical AHREs-I target genes. DIMNF (20) was also 13

found to suppress cytokine-mediated induction of the membrane complement regulatory protein CD55,

promoting tumor cells

to

complement-mediated

lysis

in

inflammatory tumor

microenvironment [125]. L-Kyn (L-Kynurenine, 21) is one of the bioactive metabolites that are formed along the kynurenine pathway of L-Trp degradation whose rate limiting step is catalyzed by IDO1 and TDO2 [126]. The discovery of L-Kyn (21) as weak ligand of AhR (Ki = 2.16x10-5 M) allowed to identify the receptor as one of the downstream effectors of immunotolerance functions associated to IDO1 and TDO2 in cancer immuno-editing process [127,128]. In this framework, the immunoregulatory signaling axis composed of IDO1/TDO2, L-Kyn (21) and AhR was also found to be pivotal in the control of disease tolerance, namely the ability of the host to reduce effects of infection states on the fitness of the host [64]. At the cellular level, interaction of L-Kyn (21) to AhR proved to stimulate generation of T(reg) cells over Th17 cells [129,130], and promote mast cell activation [131]. A comparative study of AhR-mediated transcriptional activity of L-Kyn (21) and TCDD (1) evidenced different abilities in the regulation of CYP1A1, CYP1B1, TGFβ1 and IDO1 genes in dendritic cells [83]. In particular, it was found that L-Kyn (21) mostly promotes expression of the anti-inflammatory genes TGFβ1 and IDO1, while TCDD is more effective in inducing drug-metabolizing enzymes CYP1A1 and CYP1B1. Computational studies and mutagenesis experiments also indicated a specific key interaction of L-Kyn (21) at residue Gln377 of PAS-B AhR, suggesting a mechanistic link between specific ligand binding mode and preferential transcription of anti-inflammatory genes which supports a SAhRM profile for this endogenous compound [83]. Another study reported traceextended aromatic condensation products (TEACOP270, 22 and TEACOP274, 23) from spontaneous chemical conversion of L-Kyn (21) as potent AhR agonists in COS-1 cells, suggesting L-Kyn (21) as endogenous pro-ligand of AhR [132]. The high potency of TEACOPs (22, 23) in activating AhR was instrumental to provide a tentative explanation to the high concentrations of LKyn required to activate the receptor, though formation of TEACOPs (22, 23) in vivo is still elusive. High concentrations of L-Kyn (21) to activate AhR were also explained with the presence of active 14

transport rather than passive diffusion in plasma membrane of T cells. Accordingly, the polar metabolite L-Kyn (21) is specifically transported into immune-activated T cells for receptor engagement by the System L amino acid transport pump (SLC7A5), with immune-regulated expression levels of SLC7A5 controlling AhR activation in response to L-Kyn (21) [133]. Based on the ability to promote CYP1A1 gene induction in vitro and/or in vivo, an array of approved drugs were rediscovered as AhR agonists, including nimodipine [72] (24, calcium channel blocker), flutamide (25, antiandrogen) [72], mexiletine (26, antiarrhythmic) [72], omeprazole (27, antiacid) [134], leflunomide (28, anti-inflammatory) [135], sulindac (29, anti-inflammatory) [136], tranilast (30, antiallergic drug) [137], 4-hydroxytamoxifen (31, anti-estrogenic) [138], raloxifene (32, antiestrogen) [73] and carbidopa (33, antiparkinson) [139]. Of note, some of these compounds exhibit SAhRM profile and cell-context AhR-dependent activities [140]. In particular, omeprazole (27) was among the first drugs reported as AhR agonist in literature, with such discovery dating back in ’90 [134,141]. Accordingly, in the past two decades the effects of the interaction of omeprazole (27) to AhR have been thoroughly investigated, and they are herein discussed as a case study. Although omeprazole (27) was formerly reported as weak AhR ligand [5], it has also been proposed as an atypical AhR agonist, modulating receptor functions through the activation of kinases and ensuing post-transcriptional modifications of AhR [142,143]. At this regard, mutagenesis studies of Tyr320Phe (rat sequence) and Tyr322Phe (human sequence) indicated a critical role for such kinase-substrate

residue

in

omeprazole-induced

AhR

activation

[144,145].

Chromatin

immunoprecipitation assays also revealed ligand-specific recruitment of AhR coactivators between omeprazole (27) and TCDD (1) for receptor-mediated gene expression, raising clues for a SAhRM profile of this antiacid drug [145]. Omeprazole (27) is a racemic mixture containing a chiral sulfur atom. Accordingly, enantiospecific activation of AhR was also demonstrated in cellular assays reporting the S- enantiomer as more active than the R- enantiomer at low concentrations [146]. Pharmacological studies evidenced AhR-mediated protective effects of omeprazole (27) against 15

hyperoxic lung injury in adult mice, with this effect being ascribed to induction of pulmonary CYP1A detoxifying enzymes [147]. At odds with early studies suggesting tumor promoter activity of omeprazole (27) in rat liver [148], later on findings reported the ability of omeprazole (27) to decrease breast cancer cell invasion and metastasis in vitro and in vivo [149], evidencing different tissue-specific activity of the compound. Interestingly, the molecular basis of the anticancer activity was suppression of pro-metastatic gene expression including matrix metalloproteinase-9 (MMP-9) and C-X-C chemokine receptor 4 (CXCR4), with the latter occurring through a ligand-induced and AhR-mediated mechanism. Similar anticancer effects were also observed upon treatment of invasive pancreatic cancer cells (Panc1) with omeprazole (27) [82]. In this case, inhibition of Panc1 cell invasion by omeprazole (27) was ascribed to ligand-induced activation of a non-genomic AhRmediated pathway that was dependent on Jun-N-terminal kinase (JNK) and mitogen-activated kinase-kinase 7 (MKK7) [150]. Other genomic and non-genomic AhR-mediated activities of omeprazole (27) reported in literature include stimulation of human insulin-like growth factor binding protein-1 (IGFBP-1) gene expression in hepatocarcinoma cell line (HepG2) [151], and phosphorylation of p38MAPK and cPLA2 leading to platelet activation [152].

16

Figure 5. Chemical structures of SAhRMs

17

Figure 6. Conformations of energetic minima of 3,3’-DIM (9, panel A) and its derivatives 11-13 (panels B-D). Calculations were performed using MacroModel tool of Schrödinger Software (Schrödinger Release 2019-1: MacroModel, Schrödinger, LLC, New York, NY, 2019) choosing Torsional Sampling option in the CSearch Tab and 500 as maximum number of steps.

Rapidly Metabolized AhR Ligands (RMAhRLs). The more recent term Rapidly Metabolized AhR Ligand (RMAhRL, Figure 7) refers to a receptor agonist endowed with fast metabolic degradation. It is intended to define AhR small molecule modulators that are devoid of TCDD-like toxicity because of lack of harmful bioaccumulation due to rapid elimination metabolism, and shorter lifetime of the active receptor complex they form [105]. Although they do not necessarily induce AhR-mediated transcription of selected pool of canonical and/or non-canonical target genes, RMAhRLs may include compounds that have been previously reported as SAhRMs. For instance, this is the case of β-naphthoflavone (β-NF, 34). Specifically, β-NF (34) is a positional isomer of α-NF (19) that shows strong binding affinity to AhR (EC50 = 8.4 x 10-9 M) and is also endowed with high metabolic clearance [153–155]. The activation of AhR by β-NF (34) was reported to induce expression of drug-metabolizing enzymes and be associated to immunosuppressive functions [4,72,156]. Noteworthy, a microarray gene 18

expression study in Hepa1c1c7 cells and C57BL/6 mouse liver samples evidenced ligand-specific differential gene expression between β-NF (34) and TCDD (1) [157], suggesting that compound 34 exhibits a SAhRM profile. At this regard, β-NF (34) was also found to selectively repress dystrophin Dp71 in hepatic tissue of mice [158]. In a more recent gene expression study in differentiated and undifferentiated HepaRG cell lines, β-NF (34) was reported to control AhR-mediated regulation of genes involved in metabolism of xenobiotics (CYP1A1, CYP1B1), biosynthesis of estrogens (CYP19A1), regulatory feedback loop (TIPARP), cell proliferation (STC2, SERPINB2) and cell migration capacity (ARL4C) [159]. Oral administration of β-NF in mice (34) proved to suppress pathogenesis of DSSinduced colitis without sign of toxicity, supporting a role of AhR in the immune-mediated regulation of mucosal homeostasis and inflammatory bowel disease (IBD) [160]. In another study, intraperitoneal administration of

β-NF (34) in mice proved AhR-mediated induction of liver

mitochondrial respiratory defects associated to enhanced expression and metabolic activity of CYP1A1 and CYP1A2 enzymes [161]. The binding mode of β-NF (34) to PAS-B AhR was investigated using docking studies and mutagenesis experiments [162,163]. Accordingly, it was found that Phe318 and Ile319 are key residues accounting for the interaction of 34 to PAS-B AhR. In particular, mutation of Phe318 into leucine proved to shift the activity of β-NF (34) from agonist to a partial agonist/antagonist profile similar to α-NF (19). The screening of a chemical library using a luciferase gene reporter assay was instrumental to discover 10-chloro-7H-benzimidazo[2,1-a]benzo[de]Iso-quinolin-7-one (Cl-BBQ, 35) as a high affinity ligand of AhR (IC50 = 2.6 x 10-9 M) with short half-life and no acute toxicity [164]. Structure-activity relationship studies of Cl-BBQ (35) revealed that removal of the chlorine atom (unsubstituted BBQ) or insertion of an additional chlorine atom (4,11-dichloro-BBQ) does not improve AhR transcriptional activity, while insertion of a carboxylic group, or naphthalene moiety, or 1-aminopiperidine-2,6-dione fully abolishes the activity. Additional studies proved that Cl-BBQ (35) is able to induce AhR-mediated expression of a battery of target genes similar to those 19

observed for TCDD (1) in hepatocytes and lymphocytes. Furthermore, intraperitoneal injection of Cl-BBQ (35) in mice was shown to induce T(reg) cell and suppress murine graft-versus-host disease via AhR-dependent mechanisms. Immunosuppressive activity of Cl-BBQ (35) were also investigated by oral gavage in chronic non-obese diabetic disease (NOD) murine model. Results evidenced prevention of islet infiltration without signs of toxicity, with the protective effect being not observed in AhR-deficient NOD mice [165]. Laquinimod (36) and Tasquinimod (37) are two approved drugs for the therapeutic treatment of multiple sclerosis and prostate cancer, respectively [166,167]. It was reported that the efficacy of Laquinimod (36) in arresting the clinical signs of multiple sclerosis in the experimental autoimmune encephalomyelitis (EAE) model can be ascribed to the activation of AhR signaling pathway [168]. Moreover, N-dealkylation reaction metabolism of 36 and 37 was shown to generate IMA-06201 (38) and IMA-06504 (39) metabolites that directly bind and activate AhR in vitro [169], with the former likely accounting for the AhR-mediated effects of Laquinimod (36) in the EAE model. Acetylated analogues of IMA-06201 (38) and IMA-06504 (39) were designed and synthetized (IMA-08401, 40; IMA-07101, 41) with the aim of improving their aqueous solubility and performing in vivo studies of acute and sub-acute toxicity related to AhR activity in SpragueDawley rats [170]. As a result, both compounds were able to induce AhR-mediated expression of CYP1A1 gene in all tissues without signs of toxicity, as those reported for TCDD (1). Main observed adverse effects were thymic atrophy, alterations in serum triglyceride and 3hydroxybutyrate levels, and changes in liver and kidney retinol and retinyl palmitate concentrations. In particular, elevated expression of CYP1A1 suggested a rapid elimination of the active metabolites IMA-06201 (38) and IMA-06504 (39), with this aspect being associated to the lack of TCDD-like toxicity for these compounds. In vitro toxicological studies of the active metabolites 38 and 39 further confirmed lack of cytotoxicity and genotoxicity, showing a rapid metabolic transformation of these compounds into inactive metabolites [171]. Docking studies of 38 and 39 into PAS-B AhR suggested that both ligands adopt a planar bioactive conformation with binding 20

pose similar to TCDD. Compliant to the definition of RMAhRLs, this observation supports the notion that lack of TCDD-like toxicity in 38 and 39 could be associated to fast inactivating metabolism, and not to a specific ligand binding mode in PAS-B AhR linked to a selective target gene expression. In a broader meaning of the definition, some endogenous compounds and diet-derived products or metabolites that were previously investigated as AhR agonists may also be proposed as RMAhRLs, levering the concept that these receptor ligands may have fast metabolic clearance and be non-toxic upon acute and/or chronic administration. Indole-3-carbinol (I3C, 10) is a natural compound found in cruciferous vegetables that has been studied in humans for its safety and lack of toxicity [172– 174]. Its acid-catalyzed dimerization product 3,3’-DIM (9) has been investigated in early studies as prototypical SAhRM compound [106]. Nevertheless, I3C is also able to weakly bind and activate AhR. Accordingly, the role of this receptor in the immunoregulatory and anticancer properties of I3C (10) was extensively investigated as part of the mechanism of action of this dietary compound [175–179]. In this framework, just to mention a few of such studies, dietary supplementation of I3C (10) proved to reduce morbidity and mortality associated with Clostridium difficile infection in mice through AhR-dependent and -independent mechanisms [180]. In another study, at odds with TCDD (1), dietary administration of I3C (10) in mice resulted in the enhancement of oral tolerance to dietary antigens and attenuation of peanut allergy symptoms, with AhR target engagement being demonstrated with elevated expression of CYP1A1 in intestinal tissues [181]. In contrast to L-Kyn (21), some other human metabolites of the essential dietary aminoacid L-Trp (L-Tryptophan; 42), such as ITE (2-(1’H-indole-3’-carbonyl)-thiazole-4-carboxylic acid methyl ester; 43) and FICZ (6-formylindolo[3,2-b]carbazole; 44) can be defined as RMAhRLs for their fast metabolic degradation and lack of TCDD-like toxicity. Specifically, ITE (43) is a high affinity AhR endogenous agonist (Kd = 3 x 10-9 M) that is endowed with immunosuppressive and anticancer functions [182]. It proved to have AhR-mediated induction effects on functional FoxP3+ T(reg) cells, leading to immunosuppressive effects in models of 21

experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune uveitis (EAU) [183,184]. Activation of AhR by ITE (43) was shown to inhibit Th17 cell-mediated inflammatory response, regulating dendritic cells (DCs) and CD4(+) T cells derived from patients with allergic rhinitis [185]. Anticancer activities of ITE (43) were also reported in suppressing proliferation and migration of ovarian cancer cells [186]. and reducing the tumorigenic potential of stem-like cancer cells in orthotopic xenograft tumor models [187]. Although ITE (43) and TCDD (1) were shown to provoke transcriptional activation of similar set of target genes [188], ITE (43) does not induce TCDD-like toxic effects in rat and mouse fetus [189,190]. This observation supports the classification of ITE (43) as RMAhRL, with its safety profile being ascribed to a fast metabolic elimination. Computational and mutagenesis studies also pinpointed that ITE (43) and TCDD (1) adopt similar binding modes interacting with conserved residues of PAS-B AhR. In particular, ITE (43) establishes key π-π stacking and hydrogen bond interactions with His285 and Tyr316 [191], namely two residues that are also important for TCDD (1) binding to AhR [7]. Moreover, structureactivity relationship studies with a focused library of ITE (43) analogues identified the thiazole ring of ITE (43) as key pharmacophoric element for receptor activation [191]. FICZ (44) is a UV-light oxidation product of L-Trp (42) that is formed in skin contributing to sensing environmental stimuli through activation of AhR-mediated expression of CYP1A1 and CYP1B1 in physiological and disease conditions of dermal cells [192,193]. This compound is a potent endogenous ligand of the receptor (7 x 10-11 M) and shares with TCDD (1) a similar pattern of AhR mediated expression of target genes [193], albeit quantitative differences were reported on the basis of different exposure time [194]. Computational studies and mutagenesis experiments indicated a pattern of key residues of PAS-B AhR interacting with FICZ (44) that comprise His285, Gly315, Ile319, Ser359 and Gln377 (murine sequence numbering) [8,83,132,163]. Being a good substrate of CYP1A1 and CYP1B1, FICZ (44) is rapidly metabolized into inactive products thereby forming a regulatory negative feedback loop with AhR activation that keeps low the endogenous concentrations of this ligand from potentially harmful bioaccumulation [86]. 22

Pharmacological studies demonstrated a protective role of FICZ (44) in chronic mite-induced dermatitis together with a crucial role in impairing myofibroblasts function in thyroid eye disease (TED) without affecting cell viability [195]. In another study, at odds with TCDD, FICZ (44) proved to hamper T(reg) cell development, stimulating Th17 cell differentiation and increasing sympotm severity of experimental autoimmune encephalomyelitis (EAE) in mice [90]. Treatment of chronic myeloid leukemia (CML) cells with FICZ (44) was reported to provoke a reduction of both cell numbers and clonogenic potential, supporting a role of AhR-mediated activity of FICZ (44) in

the generation of a myeloprotective phenotype [196].

Figure 7. Chemical structures of RMAhRLs

Structural and Physicochemical Properties of SAhRMs and RMAhRLs. SAhRMs and RMAhRLs indicate two classes of AhR ligands which respectively include compounds that adopt specific binding modes to the receptor with ensuing transcription of selective target genes and compounds that undergo fast inactivating metabolism with ensuing short half-life 23

of transcriptionally active AhR/ARNT heterodimer complex. However, often due to lack of pharmacokinetic data and/or thorough gene expression profile, some of these ligands may have uncertain classification, being reported in some studies as SAhRMs and in other studies as RMAhRLs. In this section we report results of a statistical analysis of structural and metabolismrelated physicochemical properties of SAhRMs and RMAhRLs, with the aim of casting lights on similarities between the two classes of AhR ligands. Specifically, a principal component analysis (PCA) using a set of 225 topological descriptors (supplementary materials, Table S1) over a dataset including 25 SAhRMs (Figure 5) and 12 RMAhRLs (Figure 7) was performed to qualitatively map these two classes of compounds in the “chemical space” as represented by the first two principal components (PC1, PC2; Figure 8). These components explain the 94.6% of the variance of the original dataset. The inspection of the score plot shows that SAhRMs and RMAhRLs do not cluster in specific areas of the chemical space, rather they include compounds with structural properties spanning a wide area of diversity.

Figure 8. Score plot of the first two components (PC1, PC2) of SAhRMs (gray circle) and RMAhRLs (black circles) performed on 225 topological descriptors. Compounds of the dataset are numbered as listed in figures 5 and 7. 24

Next, we calculated metabolism-related physicochemical properties of SAhRMs and RMAhRLs, including molecular weight (MW), octanol/water partition coefficient (XlogP3), and topological polar surface area (TPSA), using Swiss-ADME tool [197].

The statistical analysis of these

properties are reported in table 1.

Table 1. Statistical analysis of metabolism-related physicochemical descriptors

Although both SAhRMs and RMAhRLs have similar size (MW), mean and median values of calculated octanol/water partition coefficient (XlogP3) and topological polar surface area (TPSA) pinpoint high polar characteristics for RMAhRLs, with these features likely accounting for their rapid metabolic elimination. Hence, taking together the PCA study and metabolism-related physicochemical descriptors, RMAhRLs may represent a subgroup of highly polar SAhRMs.

Conclusions During the past two decades, a growing number of studies have indicated AhR as attractive drug target for the development of novel immunomodulatory agents for a therapeutic treatment of immune-related diseases. Yet, safety concerns are still associated to this receptor due to the early track record of studies linking TCDD-associated toxicity to AhR activation. In this framework, 25

some works have tried to lower safety risks of AhR ligands with the development of SAhRMs that, in contrast to TCDD (1), may induce receptor-mediated regulation of selected pool of canonical and/or non-canonical related target genes with cell- and tissue-type dependent specificity and without effects of TCDD-like toxicity. More recent medicinal chemistry efforts are attempting to develop RMAhRLs, suggesting optimization of pharmacokinetic properties of AhR lead compounds towards fast metabolic degradation as a viable strategy to avoid ligand bioaccumulation that may be likely associated to a long-acting receptor complex with ensuing TCDD-like toxic outcomes. Notwithstanding, an analysis of structural and physicochemical properties of available compounds for these two classes of ligands casts lights on common conserved structural features, suggesting that RMAhRLs may well represent a subgroup of SAhRMs endowed with high polarity that favors a fast metabolic degradation. It is our opinion that future medicinal chemistry studies of AhR ligands will likely attempt to further integrate these two concepts, tailoring SAhRMs with rapidly metabolic degradation properties. Hence, although it might still appear that the development of AhR drug candidates have high safety risks, it should be worth further investments into additional medicinal chemistry efforts, detailed structural work, in vitro and in vivo pharmacokinetic and pharmacological studies to disclose safe drug-like AhR modulators. These compounds will likely become the next-generation medicines to fight against immune-related disorders, including inflammatory and cancer diseases.

Acknowledgments This work was supported by the Italian Ministry of University and Research (PRIN 2017BZEREZ) to FF and AM. Supporting Information Available: Table S1 lists the topological descriptors and principal component loadings of the PCA study. Table S2re ports the eigenvalues and cumulative variance of the first three component of the PCA study (PC1, PC2, PC3).

26

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51

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AhR is the hub of a network linking immunity, microbiota and environment.

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AhR is now experiencing a new lease of life as target for novel immunotherapies.

•

SAhRMs are compounds that bind to AhR and activate transcription of selected target genes with specific biological outcomes.

•

RMAhRLs are agonists with specific pharmacokinetic properties leading to poor bioaccumulation and better on-target safety profile.

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SAhRMs and RMAhRLs are being pursued as next-generation medicines to fight against immune-related disorders.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: