From Tapinarof to Novel AhR Modulators: Computational Drug Discovery for Psoriasis Therapeutics

From Tapinarof to Novel AhR Modulators: Computational Drug Discovery for Psoriasis Therapeutics | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article From Tapinarof to Novel AhR Modulators: Computational Drug Discovery for Psoriasis Therapeutics Gianluca Santini, Laura Bonati, Stefano Motta This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6063129/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Jun, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor involved in the regulation of many patho-physiological processes. Among these, immune system modulation, as well as regulation of skin homeostasis and inflammation, make it a promising target for psoriasis therapy. Tapinarof, an AhR agonist recently approved for psoriasis treatment, exerts its action through antioxidant, anti-inflammatory and barrier-restoring effects. In this study, we employed a computational drug-discovery approach to identify novel AhR modulators with psoriasis therapeutic potential. We performed a multi-step similarity-based screening in PubChem. Application of molecular docking led to the identification of diverse chemical scaffolds with high docking scores and potential AhR activity, some of which belong to chemical classes with known pharmacological relevance. Notably, several identified compounds suggest a possible interplay between AhR signaling and sirtuin modulation, highlighting a previously unexplored avenue in psoriasis therapy. Our findings underscore the potential of computational approaches in accelerating the discovery of novel AhR-targeting agents and provide a foundation for further experimental validation. Biological sciences/Drug discovery/Drug screening/Virtual screening Biological sciences/Computational biology and bioinformatics/Virtual drug screening Biological sciences/Structural biology/Molecular modelling Biological sciences/Drug discovery/Medicinal chemistry/Screening Biological sciences/Drug discovery/Medicinal chemistry/Structure based drug design Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that plays a pivotal role in mediating various cellular and metabolic responses 1 – 3 . Binding to and activation of the AhR by a diverse array of exogenous and endogenous compounds lead to the induction or inhibition of diverse gene expression pathways and the production of a broad spectrum of toxic and biological effects, including cell growth, differentiation, apoptosis, and immune system modulation 4 – 8 . Initially believed to be activated only by planar, hydrophobic molecules, such as the widely studied halogenated aromatic hydrocarbons (HAH) which can elicit toxic effects 9 , AhR is now recognized as a receptor for a diverse range of environmental and endogenous ligands 10 – 12 . The effects of AhR activation may depend on the type of ligand, its concentration, and the specific binding site involved. An example of this complexity is the opposing effects induced on CD4 + T cell differentiation by the high-affinity agonists FICZ and TCDD 13 , 14 . Moreover, significant overlap between AhR ligands and those of other promiscuous receptors, such as PXR, suggests that crosstalk with other transcription factors may contribute to ligand-specific responses 15 , 16 . AhR exerts its function through both canonical and non-canonical signaling pathways 4 , 6 . The canonical AhR signaling pathway involves ligand binding to the PAS-B domain of AhR in the cytosol, where it is complexed with the chaperone heat shock protein 90 (hsp90), the co-chaperone XAP2, and the p23 protein. Hsp90 protects AhR from degradation, retains the complex within the cytoplasm, and maintains AhR in an inactive state by covering its nuclear localization sequence (NLS). Upon ligand binding, the AhR:hsp90 complex undergoes a conformational change that facilitates its translocation into the nucleus and conversion into its high-affinity DNA-binding form 17 . During this process, AhR dissociates from the chaperone proteins and dimerizes with the homologous AhR nuclear translocator (ARNT). Finally, the ligand:AhR:ARNT complex binds to a specific DNA recognition site, the dioxin-responsive element (DRE), leading to gene transcription 18 . The non-canonical functions of AhR are diverse, context-dependent, and often tissue- or ligand-specific. AhR has been found to interact with various key cellular signaling pathways that are critical for normal physiological processes, as well as in the pathogenesis of several diseases 1 , 2 , 6 . For this reason, the development of new therapeutic molecules targeting AhR is becoming increasingly promising. However, the lack of experimentally determined atomistic structures of the AhR ligand-binding domain has long hindered accurate in-silico predictions. The recent determination of the Cryo-EM structure of the AhR:hsp90:XAP2:p23 cytosolic complex marked a significant advancement 19 , enabling the use of an experimental structure as a model for in-silico investigations of AhR-ligand binding rather than relying solely on homology models 20 – 23 . Furthermore, the subsequent release of experimental AhR structures in different bound and unbound forms 24 , 25 has provided insights into ligand-induced conformational changes within the ligand-binding cavity. Notably, while we had already completed our computational analysis, a set of X-ray structures of the AhR:ARNT dimer complexed with some of the most studied AhR ligands (tapinarof, indirubin, FICZ, Benzo[a]pyrene (BaP), β-Naphthoflavone, and indigo) became available 26 . These newly resolved structures further illustrate how different ligands stabilize distinct conformations of the receptor, reinforcing our understanding of ligand-induced adaptations. Such insights enhance the accuracy of in silico modeling of AhR-ligand interactions, paving the way for more reliable computational screening approaches to identify novel AhR modulators with potential therapeutic applications. Among the potential therapeutic applications of AhR ligands, one found clinical application in May 2022, when the US FDA approved a cream containing 1% of the AhR agonist tapinarof for the topical treatment of plaque psoriasis in adults 27 . Tapinarof is a naturally derived hydroxylated stilbene (stilbenoid) produced by bacterial symbionts of entomopathogenic nematodes. Stilbenoids, such as resveratrol, are well-known for their diverse biological activities, including anti-inflammatory 28 , anti-proliferative 29 , and anti-cancer 30 effects, many of which are mediated through interactions with multiple cellular targets, including nuclear factor kappa B (NF-κB) 31 , sirtuins 32 and AhR 33 . The ability of resveratrol and its derivatives to modulate AhR activity, sometimes with contrasting effects, highlights the complexity of AhR-ligand interactions and their therapeutic potential. 33 – 38 Following the discovery of the therapeutic effect of tapinarof cream in psoriasis patients 39 – 41 , Smith et al. identified AhR as the primary target through which tapinarof exerts its efficacy in inflammatory skin conditions. 42 Tapinarof acts through multiple mechanisms, including immune regulation, skin barrier restoration, and oxidative stress reduction 43 . By activating AhR, tapinarof suppresses Th17/Th22-driven inflammation, downregulating key cytokines such as IL-17A, IL-17F, and IL-22, which are central to psoriasis pathogenesis 42 . Additionally, tapinarof promotes keratinocyte differentiation and enhances skin barrier integrity by upregulating essential structural proteins such as filaggrin and loricrin 42 . Its antioxidant properties, mediated through both direct ROS scavenging and AhR-Nrf2 pathway activation 44 , further contribute to its therapeutic efficacy. However, the fact that the effects of tapinarof are solely due to AhR activation has been debated, suggesting that additional AhR-independent mechanisms, might also contribute to its therapeutic action. 45 The aim of this work was to identify molecular scaffolds with the potential to bind and activate AhR and to exert therapeutic effects against psoriasis-related inflammation by employing computational approaches. To achieve this, we first used molecular docking to predict the binding mode of tapinarof in the human AhR PAS-B domain, identifying key interactions that stabilize its pose. Based on these insights, we implemented a multistage search strategy to systematically explore the chemical space in PubChem. To explore a broad range of potential AhR ligands, we used diverse similarity-based strategies implemented in PubChem, leading to an initial library of 19,943 compounds. The screening of these compounds by docking identified four promising chemical families with consistently high docking scores and a substructure-based expansion of these allowed to retrieve additional 77,359 molecules from PubChem. The docking results on the final set of compounds highlighted several families with optimal docking profiles and intriguing characteristics. While some exhibited structural similarity to tapinarof, differing only in minor modifications to the stilbene scaffold, others displayed significant distinctive features. A review of the relevant literature revealed that some of these compounds are implicated in biological pathways associated with psoriasis, underscoring their relevance and warranting further investigation as potential novel AhR modulators. Results Tapinarof Binding Mode Predicted by Docking To understand the molecular interactions between tapinarof and the AhR receptor, we first performed docking calculations to predict its binding mode. Our docking analysis was based on the experimental structure of the human AhR PAS-B domain in complex with indirubin (PDBID 7ZUB 19 ), ensuring a well-represented ligand-binding site. Tapinarof, an AhR agonist, exhibits a binding affinity of 100–200 nM toward the AhR ligand-binding domain 42 . The docking score obtained for tapinarof was − 10.6 kcal/mol, consistent with the redocking score of indirubin (-11.3 kcal/mol), which is expected to have a higher affinity. The predicted binding mode revealed key interactions between tapinarof and critical residues in the receptor binding cavity (Fig. 1 ). These interactions include hydrogen bonds with the side chain of Gln383 and the backbone carbonyl of Gly321, as well as aromatic π-stacking with Phe295 and His291. The predicted pose aligns well with the binding mode observed in the recently available porcine AhR X-ray structure bound to tapinarof 26 (PDB ID: 8XS6), with an RMSD of 0.6 Å between the two (Supplementary Fig. 1). Moreover, the interactions established by tapinarof are the same found for other AhR ligands, such as indirubin (Supplementary Fig. 2) and BaP 19 , 24 . Among these, the most stabilizing interaction was with Phe295, which appears to play a key role in defining the ligand orientation within the binding site. Specifically, Phe295 dictates the plane in which the ligand lies, as its aromatic side chain provides a crucial π-stacking surface. Given that most AhR ligands possess planar aromatic scaffolds, it is conceivable that they align parallel to the Phe295 side chain to establish a strong π-π interaction, which likely contributes to their binding affinity. Docking of PubChem Molecules Identified by 3D Similarity to Tapinarof To explore structurally related compounds with potential AhR activity, we performed a 3D similarity search in PubChem using tapinarof as the query (Supplementary Fig. 3). This approach identified a set of 305 candidate molecules sharing similar molecular shape and pharmacophoric features with tapinarof. Each of these compounds was then docked into the AhR ligand-binding domain to assess their potential binding affinity and interaction patterns. Of the 305 candidates, 90 compounds exhibited docking scores comparable to or better than that of tapinarof. Analysis of these compounds revealed that most exhibit only minor modifications relative to tapinarof (hereafter referred to as “tapinarof close analogues”). These modifications primarily include: hydroxylation at one or multiple positions on one or both aromatic rings; fluorination at one or multiple positions at one or both aromatic rings; and substitution of the isopropyl group with cyclopentane or cyclohexane (Supplementary Fig. 4). The binding mode of these molecules closely resembles that of tapinarof (Fig. 2 a), with additional hydroxyl groups capable of forming hydrogen bonds with residues within the binding cavity. Specifically, we observed that Ser336 and Ser346were commonly predicted to establish hydrogen bonds with these hydroxyl groups. In addition to these tapinarof close analogues, we identified three other subfamilies of compounds that consistently exhibited favorable docking scores. Condensed Rings (Fig. 2 b): This family comprises molecules in which the central double bond of the stilbene scaffold undergoes cyclization, forming a fused ring system. These rings range from simple naphthalene-like structures to five- or six-membered nitrogen- or oxygen-containing heterocycles, always forming at the unsubstituted ring of tapinarof (Supplementary Fig. 5). This cyclization enhances π-stacking interactions with Phe295, while the hydroxylated ring maintains the characteristic hydrogen bonds with the side chain of Gln383 and the backbone carbonyl of Gly321. Benzyl Addition on Central Linker (Fig. 2 c): This family includes a small number of molecules featuring a benzyl group attached to the central double bond of tapinarof (Supplementary Fig. 6). Notably, this additional ring forms an extra π-stacking interaction with Tyr322, which may contribute to increased binding stability. Aryl Addition to Isopropyl (Fig. 2 d): Compounds in this family feature an aryl ring replacing one of the methyl groups of the isopropyl moiety of tapinarof (Supplementary Fig. 7). This substitution significantly increases the molecular length, making it incompatible with the original tapinarof binding mode. Additionally, the added aryl ring exhibits considerable conformational flexibility, a characteristic uncommon among high-affinity AhR ligands. For these reasons, this subfamily appears less promising compared to the others. All these compounds maintain a high degree of similarity to tapinarof and remain structurally very close to the original molecule. As a consequence, most of them are covered by existing tapinarof patents. This limitation prompted us to expand our search beyond these close analogues, exploring alternative scaffolds that could retain strong AhR binding potential while introducing more significant structural diversity. Docking of Tapinarof Analogues: Expanding the Dataset with Structural Variants To explore a broader range of potential AhR ligands, we expanded our search in PubChem using alternative similarity-based strategies beyond the initial 3D similarity approach (see method section). This allowed us to construct a second, more diverse library comprising 19,943 compounds. The goal of this step (summarized in Supplementary Fig. 8) was to introduce greater structural variability while preserving key pharmacophoric features essential for AhR binding. Each of the compounds in the library retrieved by PubChem was subjected to docking calculations, leading to the identification of four promising chemical families characterized by consistently high docking scores: (i) tolans, analogues of stilbenoids but with triple bonds instead of the central double bond; (ii) alkene to three-membered ring cyclization, in which the central double bond is substituted by a cyclopropyl, epoxide or aziridine moiety; (iii) phenyl benzoate derivatives, featuring an ester-linked biphenyl system; (iv) alkene to imine/azo, where the central double bond is replaced by a C = N or N = N linkage. Since these families were particularly promising due to their divergence from compounds already described by the tapinarof patents, we refined our exploration within these newly identified chemical families. To achieve this, we performed a substructure-based expansion using the minimal scaffold representing each family, retrieving an additional 77,359 compounds from PubChem. This approach allowed us to systematically investigate structural variations within the most promising scaffolds and identify compounds that exhibited optimal interaction patterns. In the following section, the properties, binding modes, and interactions of the most promising compounds from the different chemical families are discussed. Tolans : This family consists of two phenyl groups attached to both ends of a -C ≡ C- (ethynyl) linker. These molecules are closely related to stilbenes as it is possible to obtain a stilbene by simple partial hydrogenation of the central triple bond. The most promising compounds here identified are hydroxylated-tolans in which at least one of the two phenyl groups is substituted with at least one -OH group. The compound with the best score (-12.45 kcal/mol) identified within this family is shown in Fig. 3 . It contains four hydroxyl groups, two on each ring. Interestingly, the positioning of these hydroxyls does not allow for the preservation of the hydrogen bond with Gly321. At the opposite end of the molecule, the hydroxyl group in the para position forms a hydrogen bond with the backbone carbonyl of Cys333. Supplementary Fig. 9 shows additional compounds belonging to the tolan family. These comprise compounds hydroxylated at different positions and molecules that feature various substituents at one of the rings or condensed rings on one side. Alkene to three-membered ring cyclization : This family consists of compounds in which the central double bond of stilbenes is replaced by a three-membered ring. Interestingly, despite the removal of the central double bond which disrupts the planarity of the molecule, the introduction of a three-membered ring confers rigidity leading to a geometry that closely resembles that of stilbenes. Notably, the two aromatic groups on opposite sides of the ring often lie on the same plane, albeit slightly offset. The compound with the highest docking score (-14.16 kcal/mol) features a cyclopropyl group at the central linker and two condensed rings (Fig. 4 ). One of these rings, an indol-2-one, forms an H-bond with Gln383 and Ser365 and is engaged in a π-stacking interaction with His291. The other ring participates in an H-bond with Ser336 and Ser346 and establishes π-stacking interactions with Phe295 and Phe351. Remarkably, this compound, identified through a search based on tapinarof, is closely related to indirubin—a well-known strong AhR activator with therapeutic potential for psoriatic disease 46 . The binding mode of this compound closely resembles that of indirubin, as seen in its experimental structure in complex with the AhR PAS-B 19 (Supplementary Fig. 10). Supplementary Fig. 11 shows additional compounds belonging to this family. Most of them feature a simple cyclization of the central stilbene double bond (forming either a cyclopropyl or an epoxide group). Interestingly, we also identified some condensed ring systems bearing similarity to well-known AhR ligands, such as BaP. Ligands with aziridine groups were found to bind with less favorable docking scores. Phenyl benzoate derivatives : This family of compounds was originally inserted into the most promising compounds, as we noted that the substitution of the stilbenes central double bond with an ester group could be a promising modification. However, after refinement of the search through the substructure function, we surprisingly discovered a high number of molecules with docking scores significantly lower than the one obtained by tapinarof, but in which the ester group form an additional condensed cycle (lactonization). Thus, the resulting molecules present three or more condensed aromatic rings. The compound with the highest docking score (-12.95 kcal/mol) presents four condensed rings that establish an extensive π-stacking network with residues Phe295 and Phe351 (Fig. 5 ). Moreover, H-bonds with Ser346, Ser336 and Gly321 anchor the molecule at opposite sides. Supplementary Fig. 12 shows additional compounds belonging to this family. Alkene to imine/azo : This family of compounds produces the largest dataset of ligands, with 58,445 compounds retrieved from PubChem. The substitution of one or both carbon atoms in the central double bond of stilbenes (as in N-benzylideneaniline and azobenzene, respectively) provides a flexible scaffold, where the central linker can accommodate an additional fused ring (a feature frequently observed in the phenyl benzoate family as well). Many compounds of this family share a 2-hydroxyquinoxaline ring, with the best compound showing a docking score of -13.84 kcal/mol. This recurrent motif forms H-bonds with Gln383 and Ser365 and engages in π-stacking with His291 (Fig. 6 ). The second ring is stabilized by π-stacking interactions with Phe295 and Phe351. Overall, the two aromatic systems lie almost in the same plane, with an interplanar angle of approximately 20°. Additional compounds from this family are shown in Supplementary Fig. 13. Discussion In this study, we employed a multistage computational drug-discovery pipeline to explore the chemical space around tapinarof, a known AhR agonist approved for psoriasis treatment. Our docking results on the tapinarof molecule align with the recent experimental data and provide a solid foundation for identifying other compounds capable of activating AhR. By conducting a 3D similarity search in PubChem, we identified 305 compounds, among which we found interesting families. However, these compounds were structurally too similar to tapinarof and were already described in the tapinarof patents. Expanding the search further, we screened 19,943 additional compounds and identified four major promising families with favorable docking scores: tolans, alkene-to-three-membered-ring cyclization, phenyl benzoate derivatives, and alkene-to-imines/azo compounds. The diverse scaffolds identified in this expanded search suggest that non-traditional structural motifs, beyond tapinarof analogues, may offer new therapeutic opportunities for psoriasis and other AhR-related diseases. Interestingly, we found data supporting further investigation into certain families of compounds. Tolans, for example, are a class of compounds already patented for their use as cosmetics or therapeutics for skin conditions 47 . Surprisingly, we discovered that the patent is related to the use of tolans as modulator of the sirtuins activity. Sirtuins are a family of enzymes, named after the yeast protein Sir2, which play a crucial role in regulating cellular processes such as aging, stress responses, and metabolism 48 . AhR and sirtuins, particularly SIRT1 and SIRT3, exhibit a bidirectional regulatory relationship with implications for skin health and inflammation 49 . AhR activation suppresses SIRT1 by reducing NAD + levels, accelerating senescence 50 , while SIRT1 enhances AhR-driven processes, including filaggrin expression 51 . SIRT1 depletion in keratinocytes has been reported to inhibit both basal and ligand-induced AhR activation, while its presence enhances AhR-driven processes, including AhR/AKT-induced filaggrin expression, which is crucial for skin barrier function 52 . Moreover, AhR activation inhibits SIRT3 via TiPARP-induced NAD + depletion, increasing oxidative stress through SOD2 acetylation 53 . The identification of tolans as AhR modulators suggests they might influence both sirtuins and AhR signaling, key players in psoriasis pathogenesis. Interestingly, tapinarof itself shares structural similarity with resveratrol, piceatannol, and other known sirtuin activators. This raises the possibility that its therapeutic effects may extend beyond AhR activation to include sirtuin modulation. Supporting this, in the work of Smith et al. 42 the molecular profiling experiment evidenced that tapinarof moderately activates SIRT1, while resveratrol—a well-established SIRT1 activator—was less potent in the same assay. Given the established crosstalk between AhR and sirtuins, further investigation is warranted to determine whether sirtuin activation contributes to tapinarof efficacy in psoriasis, potentially offering a dual mechanism of action that enhances skin barrier integrity and mitigates inflammation. This dual regulation opens new therapeutic perspectives, where targeting both pathways could help manage inflammation, oxidative stress, and skin barrier function. Other compounds also emerged as particularly noteworthy. One member of the “alkene to three-membered ring cyclization” family, Gnetumelin C (Supplementary Fig. 11a), is a naturally occurring compound known for its anti-inflammatory, antimicrobial, and antioxidant properties. It has been proposed as a promising ingredient in cosmetic formulations aimed at skin protection and anti-aging applications 54 . The top-performing compound within the “phenyl benzoate derivatives” (Fig. 5 ) shares structural similarity with ellagic acid, a naturally occurring heterotetracyclic compound found in various fruits and vegetables. Ellagic acid exhibits antioxidant and anti-proliferative effects and was studied for the topical treatments of melasma 55 . Additionally, other molecules within the “phenyl benzoate derivatives” class are particularly interesting, as their unsaturated lactone core represents the structural backbone of coumarin derivatives. These compounds are studied in inflammatory bowel diseases for their ability to activate the AhR/Nrf2 pathways. 56 , 57 Overall, our findings underscore the potential of computational drug discovery in identifying novel AhR modulators, broadening the spectrum of candidates for experimental validation. Notably, starting from the pharmacophoric features of tapinarof, our approach identified molecules with potential involvement in other psoriasis-related pathways, as well as compounds structurally similar to established AhR ligands. These results provide a strong foundation for future research and further experimental exploration. Methods PubChem Search To identify novel AhR modulators, we employed a systematic multi-stage ligand search strategy using the PubChem database 58 . PubChem provides several tools for ligand-based searching, including 2D similarity search, 3D similarity search, and substructure search. The multi-stage search strategy is schematized in Supplementary Fig. 3 and Supplementary Fig. 8. The 2D similarity search in PubChem relies on molecular fingerprints, which encode the presence or absence of specific substructural patterns within a molecule. The search algorithm computes a similarity score, typically using the Tanimoto coefficient, to compare the query molecule with the compounds in the database. This approach enables the retrieval of structurally related compounds that share common functional groups and topological features with the query molecule. For our study, we used the PubChem 2D similarity search tool in the second step of the multi-stage search strategy, querying against tapinarof, with the default Tanimoto threshold of 0.9. Unlike 2D searches, which rely on molecular connectivity, 3D similarity searches evaluate the spatial conformation of molecules. PubChem's 3D search algorithm compares the three-dimensional shape and electrostatic properties of a query molecule against the conformers stored in its database. The scoring function ranks molecules based on shape similarity and pharmacophoric match, providing a set of molecules that may preserve binding modes of the reference ligand. For our study, we used the PubChem 3D similarity search tool in both the first and second steps of the multi-stage search strategy. In the first step, we queried against tapinarof analogs, and in the second step, we expanded the search by querying against 1,3-dichloro-5-(2-phenylethenyl)benzene. The choice of chlorinated trans-stilbene as the target for the 3D similarity search was made because, despite non-hydroxylated trans-stilbenes are known to be good binders of AhR, they were not retrieved in the first 3D similarity search against tapinarof, which predominantly returned molecules with one or more hydrogen donor groups. The substructure search tool in PubChem identifies compounds containing a specific molecular core scaffold. This approach is useful for exploring chemical families that share the desired scaffold with the reference compound while allowing for significant variations in the functional groups. To expand our search space, we used the substructure search based on key scaffolds identified in the second step of the multi-stage search. Given that the substructure search may return molecules that differ significantly from the original compound, as long as they contain the searched substructure, we applied filters on size (molecular weight < 300 g/mol) and hydrophobicity ( -1 < logP < 5) to reduce the number of compounds obtained from the search, while remaining close to the queried ligand. The filter on hydrophobicity was not applied to the family of compounds with three-membered ring substitution of the central double bond, as the number of compounds retrieved for this family was already small. Molecular Docking The structure of the human AhR PAS-B domain (PDB ID: 7ZUB 19 ) was obtained from the Protein Data Bank. This Cryo-EM structure includes the AhR PAS-B domain complexed with the indirubin ligand and the hsp90 and XAP2 proteins that constitute its cytosolic assembly. For docking calculations, non-AhR proteins were removed, and the resulting structure was preprocessed using Schrödinger’s Protein Preparation Wizard. 59 Residue protonation states were assigned with PROPKA 60 at pH 7.0. The structures of the ligands were downloaded from PubChem in the sdf format and then prepared with the LigPrep utility in the Schrödinger 2024-2 suite 61 . Their protonation states were determined with the Epik Classic tool for pKa prediction included in Maestro 62 , that is based on PROPKA as heuristic pKa calculator. Docking was performed using Glide XP 63 (extra precision). This method uses a hierarchical series of filters to search for possible locations of the ligand in the binding site and includes a flexible treatment of the ligand. The shape and properties of the protein are represented on a grid by different sets of fields that provide progressively more accurate scoring of the ligand poses. Glide XP performs extensive sampling for ligand positioning through an anchor-and-grow approach and also accounts for explicit waters. The method uses a scoring function (XP GlideScore) that includes force-field-based functions to describe Coulomb and van der Waals contributions to the interaction energy as well as empirically based functions. The receptor grid for the AhR PAS-B domain was centered on the center of mass of the indirubin ligand from the experimental structure. Docking calculations employed the following parameters: a) Retain up to 50,000 poses per ligand during the initial docking phase. b) Use a scoring window of 200 kcal/mol to select initial poses. c) Retain up to 2,000 poses per ligand for energy minimization. d) Apply expanded sampling to increase thoroughness. Ligands Cluster Analysis To analyze the results of the virtual screening for the large dataset of ligands obtained in stage 2, we performed a cluster analysis of the ligands that displayed a docking score lower than − 9.5 kcal/mol. This threshold was chosen to include only ligands with docking scores within 1 kcal/mol of the score obtained by tapinarof. The subset of ligands meeting this criterion was clustered using the Canvas Similarity and Clustering tool in Maestro. We used a linear fingerprint type with the atom typing scheme 12 (Daylight invariant atom types, where bonds are distinguished by bond order, and cyclic aliphatic structures are distinguished from acyclic aliphatic ones). Similarity was computed using the Tanimoto similarity metric, and results were then clustered using the complete linkage method. The optimal number of clusters was determined based on the Kelley Penalty score. Clusters were analyzed to identify families of ligands that displayed consistently favorable docking scores. Declarations Author contributions statement S.M and L.B. conceived the presented idea, G.S. performed the computations and analyzed the results, S.M supervised the project, L.B. helped supervise the project, G.S. aided in interpreting the results, S.M. drafted the manuscript and designed the figures. All authors discussed the results and reviewed the manuscript. Additional information Competing interests: The authors declare no competing interests. Author Contribution S.M and L.B. conceived the presented idea, G.S. performed the computations and analyzed the results, S.M supervised the project, L.B. helped supervise the project, G.S. aided in interpreting the results, S.M. drafted the manuscript and designed the figures. All authors discussed the results and reviewed the manuscript. Acknowledgement This research was supported by the National Psoriasis Foundation USA (Discovery Grant - Award ID: 1298983). Data Availability The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. References Larigot, L., Juricek, L., Dairou, J. & Coumoul, X. AhR signaling pathways and regulatory functions. Biochim Open 7 , 1–9 (2018). Esser, C. & Rannug, A. The aryl hydrocarbon receptor in barrier organ physiology, immunology, and toxicology. Pharmacol Rev 67 , 259–279 (2015). Murray, I. A. & Perdew, G. H. How Ah Receptor Ligand Specificity Became Important in Understanding Its Physiological Function. Int J Mol Sci 21 , 9614 (2020). Wright, E. J., Pereira De Castro, K., Joshi, A. D. & Elferink, C. J. Canonical and non-canonical aryl hydrocarbon receptor signaling pathways. Curr Opin Toxicol 2 , 87–92 (2017). Riaz, F., Pan, F. & Wei, P. 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S. & Bonati, L. Structural modeling of the AhR:ARNT complex in the bHLH-PASA-PASB region elucidates the key determinants of dimerization. Mol Biosyst 13 , 981–990 (2017). Pandini, A., Denison, M. S., Song, Y., Soshilov, A. A. & Bonati, L. Structural and functional characterization of the aryl hydrocarbon receptor ligand binding domain by homology modeling and mutational analysis. Biochemistry 46 , 696–708 (2007). Kwong, H. S. et al. Structural Insights into the Activation of Human Aryl Hydrocarbon Receptor by the Environmental Contaminant Benzo[a]pyrene and Structurally Related Compounds. J Mol Biol 436 , (2024). Wen, Z. et al. Cryo-EM structure of the cytosolic AhR complex. Structure 31 , 295-308.e4 (2023). Diao, X. et al. Structural basis for the ligand-dependent activation of heterodimeric AHR-ARNT complex. Nat Commun 16 , 1282 (2025). Keam, S. J. Tapinarof Cream 1%: First Approval. Drugs 82 , 1221–1228 (2022). Rangarajan, P., Karthikeyan, A. & Dheen, S. T. Role of dietary phenols in mitigating microglia-mediated neuroinflammation. Neuromolecular Med 18 , 453–464 (2016). De Leo, A., Arena, G., Stecca, C., Raciti, M. & Mattia, E. Resveratrol Inhibits Proliferation and Survival of Epstein Barr Virus–Infected Burkitt’s Lymphoma Cells Depending on Viral Latency Program. Molecular Cancer Research 9 , 1346–1355 (2011). Chimento, A. et al. Resveratrol and Its Analogs As Antitumoral Agents For Breast Cancer Treatment. Mini-Reviews in Medicinal Chemistry 16 , 699–709 (2016). Kubota, S. et al. Prevention of Ocular Inflammation in Endotoxin-Induced Uveitis with Resveratrol by Inhibiting Oxidative Damage and Nuclear Factor–κB Activation. Investigative Opthalmology & Visual Science 50 , 3512 (2009). Howitz, K. T. et al. Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature 425 , 191–196 (2003). Mohammadi-Bardbori, A., Bengtsson, J., Rannug, U., Rannug, A. & Wincent, E. Quercetin, Resveratrol, and Curcumin Are Indirect Activators of the Aryl Hydrocarbon Receptor (AHR). Chem Res Toxicol 25 , 1878–1884 (2012). Gouédard, C., Barouki, R. & Morel, Y. Induction of the Paraoxonase-1 Gene Expression by Resveratrol. Arterioscler Thromb Vasc Biol 24 , 2378–2383 (2004). Lee, J.-E. & Safe, S. Involvement of a post-transcriptional mechanism in the inhibition of CYP1A1 expression by resveratrol in breast cancer cells. Biochem Pharmacol 62 , 1113–1124 (2001). Casper, R. F. et al. Resveratrol Has Antagonist Activity on the Aryl Hydrocarbon Receptor: Implications for Prevention of Dioxin Toxicity. Mol Pharmacol 56 , 784–790 (1999). Ciolino, H. P., Daschner, P. J. & Yeh, G. C. Resveratrol inhibits transcription of CYP1A1 in vitro by preventing activation of the aryl hydrocarbon receptor. Cancer Res 58 , 5707–12 (1998). Pastorková, B., Vrzalová, A., Bachleda, P. & Dvořák, Z. Hydroxystilbenes and methoxystilbenes activate human aryl hydrocarbon receptor and induce CYP1A genes in human hepatoma cells and human hepatocytes. Food and Chemical Toxicology 103 , 122–132 (2017). Bissonnette, R. et al. Efficacy and safety of topical WBI-1001 in patients with mild to severe atopic dermatitis: results from a 12-week, multicentre, randomized, placebo-controlled double-blind trial. British Journal of Dermatology 166 , 853–860 (2012). Bissonnette, R. et al. Efficacy and safety of topical WBI‐1001 in patients with mild to moderate psoriasis: results from a randomized double‐blind placebo‐controlled, phase II trial. Journal of the European Academy of Dermatology and Venereology 26 , 1516–1521 (2012). Bissonnette, R. et al. Efficacy and Safety of Topical WBI-1001 in the Treatment of Atopic Dermatitis: Results From a Phase 2A, Randomized, Placebo-Controlled Clinical Trial. Arch Dermatol 146 , (2010). Smith, S. H. et al. Tapinarof Is a Natural AhR Agonist that Resolves Skin Inflammation in Mice and Humans. Journal of Investigative Dermatology 137 , 2110–2119 (2017). Bissonnette, R., Stein Gold, L., Rubenstein, D. S., Tallman, A. M. & Armstrong, A. Tapinarof in the treatment of psoriasis: A review of the unique mechanism of action of a novel therapeutic aryl hydrocarbon receptor–modulating agent. J Am Acad Dermatol 84 , 1059–1067 (2021). Furue, M. et al. Antioxidants for Healthy Skin: The Emerging Role of Aryl Hydrocarbon Receptors and Nuclear Factor-Erythroid 2-Related Factor-2. Nutrients 9 , 223 (2017). Haarmann-Stemmann, T., Sutter, T. R., Krutmann, J. & Esser, C. The mode of action of tapinarof may not only depend on the activation of cutaneous aryl hydrocarbon receptor signaling but also on its antimicrobial activity. J Am Acad Dermatol 85 , e33–e34 (2021). Xie, X. et al. Indirubin ameliorates imiquimod-induced psoriasis-like skin lesions in mice by inhibiting inflammatory responses mediated by IL-17A-producing γδ T cells. Mol Immunol 101 , 386–395 (2018). Tsai, C.-C. Hydroxylated tolans and related compounds as cosmetics or therapeutics for skin conditions. (2009). Preyat, N. & Leo, O. Sirtuin deacylases: a molecular link between metabolism and immunity. J Leukoc Biol 93 , 669–680 (2013). Mulero-Navarro, S. & Fernandez-Salguero, P. M. New Trends in Aryl Hydrocarbon Receptor Biology. Front Cell Dev Biol 4 , (2016). Koizumi, M. et al. Aryl Hydrocarbon Receptor Mediates Indoxyl Sulfate-Induced Cellular Senescence in Human Umbilical Vein Endothelial Cells. J Atheroscler Thromb 21 , 904–916 (2014). van den Bogaard, E. H. et al. Genetic and Pharmacological Analysis Identifies a Physiological Role for the AHR in Epidermal Differentiation. Journal of Investigative Dermatology 135 , 1320–1328 (2015). Ming, M. et al. Loss of sirtuin 1 (SIRT1) disrupts skin barrier integrity and sensitizes mice to epicutaneous allergen challenge. Journal of Allergy and Clinical Immunology 135 , 936-945.e4 (2015). He, J. et al. Activation of the Aryl Hydrocarbon Receptor Sensitizes Mice to Nonalcoholic Steatohepatitis by Deactivating Mitochondrial Sirtuin Deacetylase Sirt3. Mol Cell Biol 33 , 2047–2055 (2013). Gnetumelin C. https://www.smolecule.com/products/s13573037 . Sivamani, R. & Clark, A. Phytochemicals in the treatment of hyperpigmentation. Botanics Volume 6 , 89–96 (2016). Singh, R. et al. Enhancement of the gut barrier integrity by a microbial metabolite through the Nrf2 pathway. Nat Commun 10 , 89 (2019). Di Stasi, L. C. Natural Coumarin Derivatives Activating Nrf2 Signaling Pathway as Lead Compounds for the Design and Synthesis of Intestinal Anti-Inflammatory Drugs. Pharmaceuticals 16 , 511 (2023). Kim, S. et al. PubChem 2025 update. Nucleic Acids Res 53 , D1516–D1525 (2025). Madhavi Sastry, G. et al. Protein and ligand preparation: Parameters, protocols, and influence on virtual screening enrichments. J. Comput. Aid. Mol. Des 27 , 221–34 (2013). Søndergaard, C. R., Olsson, M. H. M., Rostkowski, M. & Jensen, J. H. Improved Treatment of Ligands and Coupling Effects in Empirical Calculation and Rationalization of p Ka Values. J Chem Theory Comput 7 , 2284–2295 (2011). Greenwood, J. R., Calkins, D., Sullivan, A. P. & Shelley, J. C. Towards the comprehensive, rapid, and accurate prediction of the favorable tautomeric states of drug-like molecules in aqueous solution. Journal of Computer-Aided Molecular Design vol. 24 591–604 Preprint at https://doi.org/10.1007/s10822-010-9349-1 (2010). Shelley, J. C. et al. Epik: A software program for pKa prediction and protonation state generation for drug-like molecules. J Comput Aided Mol Des 21 , 681–691 (2007). Friesner, R. A. et al. Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes. J Med Chem 49 , 6177–96 (2006). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6063129","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":418828766,"identity":"644480ba-cf21-4732-b409-d4ae43881e42","order_by":0,"name":"Gianluca Santini","email":"","orcid":"","institution":"University of Milano-Bicocca","correspondingAuthor":false,"prefix":"","firstName":"Gianluca","middleName":"","lastName":"Santini","suffix":""},{"id":418828767,"identity":"a200a157-9f8a-4e19-81f9-9af693a09981","order_by":1,"name":"Laura Bonati","email":"","orcid":"","institution":"University of Milano-Bicocca","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"","lastName":"Bonati","suffix":""},{"id":418828768,"identity":"4e17ca05-8ee5-4aca-8598-551be02480e1","order_by":2,"name":"Stefano Motta","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIiWNgGAWjYJCCDwhmBQMDGwNjGwODAR71QBUzICxmxgaGM3AtePSgaAEpBovhs0Z+fvPBhp87bBh02/uPP/g573Aen9jhtgcMBX9wajE4xpbY2HsmjcHszGHGxt5th4vZpBPbDfA5zICNx/wBb9thBrMbyYwNvNsOJ7ZJJ7ZJ4NMi38b/sfEvVEvj3zlEaGE4xsPYDLOlmbeBCC0Gx9IMm2Xb0niAfjGcLXMsHaSl3SDBwBi3w5oPP2x822YjZ3a88cHHNzXWifNnpz978OGPHG6HQQEPKjeBoIZRMApGwSgYBfgAALBBVEhtEQ6HAAAAAElFTkSuQmCC","orcid":"","institution":"University of Milano-Bicocca","correspondingAuthor":true,"prefix":"","firstName":"Stefano","middleName":"","lastName":"Motta","suffix":""}],"badges":[],"createdAt":"2025-02-19 10:23:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6063129/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6063129/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-03626-z","type":"published","date":"2025-06-06T15:56:56+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":76875126,"identity":"ff48f4e5-6adf-4c1a-93f4-fa9a0a5fb762","added_by":"auto","created_at":"2025-02-21 16:03:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":908891,"visible":true,"origin":"","legend":"\u003cp\u003eBinding mode of the tapinarof molecule within the AhR PAS-B domain. a. 3D representation of the binding pose: tapinarof is depicted in yellow sticks; protein is shown as white cartoon; relevant residues are represented in white sticks; π-stacking interactions are shown with green dashed lines; H-bonds are represented as magenta dashed lines. b. 2D representation of the tapinarof interactions.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6063129/v1/53776ce4e17db7f313f0cb15.png"},{"id":76873496,"identity":"3da194f4-5093-402a-878f-bf6c443f53ae","added_by":"auto","created_at":"2025-02-21 15:47:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3028755,"visible":true,"origin":"","legend":"\u003cp\u003eBinding mode of the most promising scaffolds identified through a 3D similarity search using tapinarof as the query molecule. For each family, the compound with the best docking score is shown. a. tapinarof close analogues; b. condensed rings; c. benzyl addition on central linker; d. aryl addition to isopropyl. 2D representation of the ligand interactions are shown in the left panels, while 3D representations of the binding pose are shown in the right panels.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6063129/v1/fdba801986f4d09c02384fd7.png"},{"id":76873498,"identity":"4746e6cd-36a1-414c-b5e3-6c29e31d9b5f","added_by":"auto","created_at":"2025-02-21 15:47:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":946555,"visible":true,"origin":"","legend":"\u003cp\u003eBinding mode of the compound with the best docking score belonging to the tolan family. a. 3D representation of the binding pose: ligand is depicted in yellow sticks; protein is shown as white cartoon; relevant residues are represented in white sticks; π-stacking interactions are shown with green dashed lines; H-bonds are represented as magenta dashed lines. b. 2D representation of the ligand interactions.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6063129/v1/0519cb074d1b189ed3d7be17.png"},{"id":76873497,"identity":"b7b70a5b-4dc2-4aa6-8515-3b6bf5b76672","added_by":"auto","created_at":"2025-02-21 15:47:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":894637,"visible":true,"origin":"","legend":"\u003cp\u003eBinding mode of the compound with the best docking score belonging to the “alkene to three-membered ring cyclization” family. a. 3D representation of the binding pose: ligand is depicted in yellow sticks; protein is shown as white cartoon; relevant residues are represented in white sticks; π-stacking interactions are shown with green dashed lines; H-bonds are represented as magenta dashed lines. b. 2D representation of the ligand interactions.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6063129/v1/3ff98daf7c519238f71f1fb8.png"},{"id":76874458,"identity":"1e63da92-6e5b-4581-ae7c-b8f8ad0bc3ea","added_by":"auto","created_at":"2025-02-21 15:55:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":912030,"visible":true,"origin":"","legend":"\u003cp\u003eBinding mode of the compound with the best docking score belonging to the “phenyl benzoate derivatives” family. a. 3D representation of the binding pose: ligand is depicted in yellow sticks; protein is shown as white cartoon; relevant residues are represented in white sticks; π-stacking interactions are shown with green dashed lines; H-bonds are represented as magenta dashed lines. b. 2D representation of the ligand interactions.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6063129/v1/c675d4a0fef2fa4836632fc1.png"},{"id":76874461,"identity":"72737445-2412-4313-95d9-24c2cb944afc","added_by":"auto","created_at":"2025-02-21 15:55:38","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":859900,"visible":true,"origin":"","legend":"\u003cp\u003eBinding mode of the compound with the best docking score belonging to the “alkene to imine/azo” family. a. 3D representation of the binding pose: ligand is depicted in yellow sticks; protein is shown as white cartoon; relevant residues are represented in white sticks; π-stacking interactions are shown with green dashed lines; H-bonds are represented as magenta dashed lines. b. 2D representation of the ligand interactions.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6063129/v1/877a911084830bec0bdadf63.png"},{"id":84242508,"identity":"3d2e84ba-11c8-4a87-aae4-5966de4fc579","added_by":"auto","created_at":"2025-06-09 16:08:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8327590,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6063129/v1/f4f91298-c556-421b-883d-d945e98ad126.pdf"},{"id":76873495,"identity":"56638317-a164-43ec-b72e-cb87a6ef9df1","added_by":"auto","created_at":"2025-02-21 15:47:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":4453428,"visible":true,"origin":"","legend":"","description":"","filename":"GSantiniSI.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6063129/v1/ea66b52c3d87d2c9ff1e4614.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"From Tapinarof to Novel AhR Modulators: Computational Drug Discovery for Psoriasis Therapeutics","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that plays a pivotal role in mediating various cellular and metabolic responses\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Binding to and activation of the AhR by a diverse array of exogenous and endogenous compounds lead to the induction or inhibition of diverse gene expression pathways and the production of a broad spectrum of toxic and biological effects, including cell growth, differentiation, apoptosis, and immune system modulation\u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6 CR7\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Initially believed to be activated only by planar, hydrophobic molecules, such as the widely studied halogenated aromatic hydrocarbons (HAH) which can elicit toxic effects\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, AhR is now recognized as a receptor for a diverse range of environmental and endogenous ligands\u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The effects of AhR activation may depend on the type of ligand, its concentration, and the specific binding site involved. An example of this complexity is the opposing effects induced on CD4\u003csup\u003e+\u003c/sup\u003e T cell differentiation by the high-affinity agonists FICZ and TCDD\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Moreover, significant overlap between AhR ligands and those of other promiscuous receptors, such as PXR, suggests that crosstalk with other transcription factors may contribute to ligand-specific responses\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAhR exerts its function through both canonical and non-canonical signaling pathways\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The canonical AhR signaling pathway involves ligand binding to the PAS-B domain of AhR in the cytosol, where it is complexed with the chaperone heat shock protein 90 (hsp90), the co-chaperone XAP2, and the p23 protein. Hsp90 protects AhR from degradation, retains the complex within the cytoplasm, and maintains AhR in an inactive state by covering its nuclear localization sequence (NLS). Upon ligand binding, the AhR:hsp90 complex undergoes a conformational change that facilitates its translocation into the nucleus and conversion into its high-affinity DNA-binding form\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. During this process, AhR dissociates from the chaperone proteins and dimerizes with the homologous AhR nuclear translocator (ARNT). Finally, the ligand:AhR:ARNT complex binds to a specific DNA recognition site, the dioxin-responsive element (DRE), leading to gene transcription\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The non-canonical functions of AhR are diverse, context-dependent, and often tissue- or ligand-specific. AhR has been found to interact with various key cellular signaling pathways that are critical for normal physiological processes, as well as in the pathogenesis of several diseases\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. For this reason, the development of new therapeutic molecules targeting AhR is becoming increasingly promising. However, the lack of experimentally determined atomistic structures of the AhR ligand-binding domain has long hindered accurate \u003cem\u003ein-silico\u003c/em\u003e predictions. The recent determination of the Cryo-EM structure of the AhR:hsp90:XAP2:p23 cytosolic complex marked a significant advancement\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, enabling the use of an experimental structure as a model for \u003cem\u003ein-silico\u003c/em\u003e investigations of AhR-ligand binding rather than relying solely on homology models\u003csup\u003e\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Furthermore, the subsequent release of experimental AhR structures in different bound and unbound forms\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e has provided insights into ligand-induced conformational changes within the ligand-binding cavity. Notably, while we had already completed our computational analysis, a set of X-ray structures of the AhR:ARNT dimer complexed with some of the most studied AhR ligands (tapinarof, indirubin, FICZ, Benzo[a]pyrene (BaP), β-Naphthoflavone, and indigo) became available\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. These newly resolved structures further illustrate how different ligands stabilize distinct conformations of the receptor, reinforcing our understanding of ligand-induced adaptations. Such insights enhance the accuracy of in silico modeling of AhR-ligand interactions, paving the way for more reliable computational screening approaches to identify novel AhR modulators with potential therapeutic applications.\u003c/p\u003e \u003cp\u003eAmong the potential therapeutic applications of AhR ligands, one found clinical application in May 2022, when the US FDA approved a cream containing 1% of the AhR agonist tapinarof for the topical treatment of plaque psoriasis in adults\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Tapinarof is a naturally derived hydroxylated stilbene (stilbenoid) produced by bacterial symbionts of entomopathogenic nematodes. Stilbenoids, such as resveratrol, are well-known for their diverse biological activities, including anti-inflammatory\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, anti-proliferative\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, and anti-cancer\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e effects, many of which are mediated through interactions with multiple cellular targets, including nuclear factor kappa B (NF-κB)\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, sirtuins\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e and AhR\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. The ability of resveratrol and its derivatives to modulate AhR activity, sometimes with contrasting effects, highlights the complexity of AhR-ligand interactions and their therapeutic potential.\u003csup\u003e\u003cspan additionalcitationids=\"CR34 CR35 CR36 CR37\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e Following the discovery of the therapeutic effect of tapinarof cream in psoriasis patients\u003csup\u003e\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, Smith et al. identified AhR as the primary target through which tapinarof exerts its efficacy in inflammatory skin conditions.\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e Tapinarof acts through multiple mechanisms, including immune regulation, skin barrier restoration, and oxidative stress reduction\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. By activating AhR, tapinarof suppresses Th17/Th22-driven inflammation, downregulating key cytokines such as IL-17A, IL-17F, and IL-22, which are central to psoriasis pathogenesis\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Additionally, tapinarof promotes keratinocyte differentiation and enhances skin barrier integrity by upregulating essential structural proteins such as filaggrin and loricrin\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Its antioxidant properties, mediated through both direct ROS scavenging and AhR-Nrf2 pathway activation\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, further contribute to its therapeutic efficacy. However, the fact that the effects of tapinarof are solely due to AhR activation has been debated, suggesting that additional AhR-independent mechanisms, might also contribute to its therapeutic action.\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe aim of this work was to identify molecular scaffolds with the potential to bind and activate AhR and to exert therapeutic effects against psoriasis-related inflammation by employing computational approaches. To achieve this, we first used molecular docking to predict the binding mode of tapinarof in the human AhR PAS-B domain, identifying key interactions that stabilize its pose. Based on these insights, we implemented a multistage search strategy to systematically explore the chemical space in PubChem. To explore a broad range of potential AhR ligands, we used diverse similarity-based strategies implemented in PubChem, leading to an initial library of 19,943 compounds. The screening of these compounds by docking identified four promising chemical families with consistently high docking scores and a substructure-based expansion of these allowed to retrieve additional 77,359 molecules from PubChem. The docking results on the final set of compounds highlighted several families with optimal docking profiles and intriguing characteristics. While some exhibited structural similarity to tapinarof, differing only in minor modifications to the stilbene scaffold, others displayed significant distinctive features. A review of the relevant literature revealed that some of these compounds are implicated in biological pathways associated with psoriasis, underscoring their relevance and warranting further investigation as potential novel AhR modulators.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eTapinarof Binding Mode Predicted by Docking\u003c/h2\u003e \u003cp\u003eTo understand the molecular interactions between tapinarof and the AhR receptor, we first performed docking calculations to predict its binding mode. Our docking analysis was based on the experimental structure of the human AhR PAS-B domain in complex with indirubin (PDBID 7ZUB\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e), ensuring a well-represented ligand-binding site. Tapinarof, an AhR agonist, exhibits a binding affinity of 100\u0026ndash;200 nM toward the AhR ligand-binding domain\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. The docking score obtained for tapinarof was \u0026minus;\u0026thinsp;10.6 kcal/mol, consistent with the redocking score of indirubin (-11.3 kcal/mol), which is expected to have a higher affinity. The predicted binding mode revealed key interactions between tapinarof and critical residues in the receptor binding cavity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese interactions include hydrogen bonds with the side chain of Gln383 and the backbone carbonyl of Gly321, as well as aromatic π-stacking with Phe295 and His291. The predicted pose aligns well with the binding mode observed in the recently available porcine AhR X-ray structure bound to tapinarof\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e (PDB ID: 8XS6), with an RMSD of 0.6 \u0026Aring; between the two (Supplementary Fig.\u0026nbsp;1). Moreover, the interactions established by tapinarof are the same found for other AhR ligands, such as indirubin (Supplementary Fig.\u0026nbsp;2) and BaP \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Among these, the most stabilizing interaction was with Phe295, which appears to play a key role in defining the ligand orientation within the binding site. Specifically, Phe295 dictates the plane in which the ligand lies, as its aromatic side chain provides a crucial π-stacking surface. Given that most AhR ligands possess planar aromatic scaffolds, it is conceivable that they align parallel to the Phe295 side chain to establish a strong π-π interaction, which likely contributes to their binding affinity.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDocking of PubChem Molecules Identified by 3D Similarity to Tapinarof\u003c/h3\u003e\n\u003cp\u003eTo explore structurally related compounds with potential AhR activity, we performed a 3D similarity search in PubChem using tapinarof as the query (Supplementary Fig.\u0026nbsp;3). This approach identified a set of 305 candidate molecules sharing similar molecular shape and pharmacophoric features with tapinarof. Each of these compounds was then docked into the AhR ligand-binding domain to assess their potential binding affinity and interaction patterns. Of the 305 candidates, 90 compounds exhibited docking scores comparable to or better than that of tapinarof. Analysis of these compounds revealed that most exhibit only minor modifications relative to tapinarof (hereafter referred to as \u0026ldquo;tapinarof close analogues\u0026rdquo;). These modifications primarily include: hydroxylation at one or multiple positions on one or both aromatic rings; fluorination at one or multiple positions at one or both aromatic rings; and substitution of the isopropyl group with cyclopentane or cyclohexane (Supplementary Fig.\u0026nbsp;4). The binding mode of these molecules closely resembles that of tapinarof (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), with additional hydroxyl groups capable of forming hydrogen bonds with residues within the binding cavity. Specifically, we observed that Ser336 and Ser346were commonly predicted to establish hydrogen bonds with these hydroxyl groups.\u003c/p\u003e \u003cp\u003eIn addition to these tapinarof close analogues, we identified three other subfamilies of compounds that consistently exhibited favorable docking scores.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eCondensed Rings\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb): This family comprises molecules in which the central double bond of the stilbene scaffold undergoes cyclization, forming a fused ring system. These rings range from simple naphthalene-like structures to five- or six-membered nitrogen- or oxygen-containing heterocycles, always forming at the unsubstituted ring of tapinarof (Supplementary Fig.\u0026nbsp;5). This cyclization enhances π-stacking interactions with Phe295, while the hydroxylated ring maintains the characteristic hydrogen bonds with the side chain of Gln383 and the backbone carbonyl of Gly321.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eBenzyl Addition on Central Linker\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec): This family includes a small number of molecules featuring a benzyl group attached to the central double bond of tapinarof (Supplementary Fig.\u0026nbsp;6). Notably, this additional ring forms an extra π-stacking interaction with Tyr322, which may contribute to increased binding stability.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eAryl Addition to Isopropyl\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed): Compounds in this family feature an aryl ring replacing one of the methyl groups of the isopropyl moiety of tapinarof (Supplementary Fig.\u0026nbsp;7). This substitution significantly increases the molecular length, making it incompatible with the original tapinarof binding mode. Additionally, the added aryl ring exhibits considerable conformational flexibility, a characteristic uncommon among high-affinity AhR ligands. For these reasons, this subfamily appears less promising compared to the others.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eAll these compounds maintain a high degree of similarity to tapinarof and remain structurally very close to the original molecule. As a consequence, most of them are covered by existing tapinarof patents. This limitation prompted us to expand our search beyond these close analogues, exploring alternative scaffolds that could retain strong AhR binding potential while introducing more significant structural diversity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eDocking of Tapinarof Analogues: Expanding the Dataset with Structural Variants\u003c/h3\u003e\n\u003cp\u003eTo explore a broader range of potential AhR ligands, we expanded our search in PubChem using alternative similarity-based strategies beyond the initial 3D similarity approach (see method section). This allowed us to construct a second, more diverse library comprising 19,943 compounds. The goal of this step (summarized in Supplementary Fig.\u0026nbsp;8) was to introduce greater structural variability while preserving key pharmacophoric features essential for AhR binding. Each of the compounds in the library retrieved by PubChem was subjected to docking calculations, leading to the identification of four promising chemical families characterized by consistently high docking scores: (i) tolans, analogues of stilbenoids but with triple bonds instead of the central double bond; (ii) alkene to three-membered ring cyclization, in which the central double bond is substituted by a cyclopropyl, epoxide or aziridine moiety; (iii) phenyl benzoate derivatives, featuring an ester-linked biphenyl system; (iv) alkene to imine/azo, where the central double bond is replaced by a C\u0026thinsp;=\u0026thinsp;N or N\u0026thinsp;=\u0026thinsp;N linkage.\u003c/p\u003e \u003cp\u003eSince these families were particularly promising due to their divergence from compounds already described by the tapinarof patents, we refined our exploration within these newly identified chemical families. To achieve this, we performed a substructure-based expansion using the minimal scaffold representing each family, retrieving an additional 77,359 compounds from PubChem. This approach allowed us to systematically investigate structural variations within the most promising scaffolds and identify compounds that exhibited optimal interaction patterns. In the following section, the properties, binding modes, and interactions of the most promising compounds from the different chemical families are discussed.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eTolans\u003c/b\u003e: This family consists of two phenyl groups attached to both ends of a -C\u0026thinsp;\u0026equiv;\u0026thinsp;C- (ethynyl) linker. These molecules are closely related to stilbenes as it is possible to obtain a stilbene by simple partial hydrogenation of the central triple bond. The most promising compounds here identified are hydroxylated-tolans in which at least one of the two phenyl groups is substituted with at least one -OH group. The compound with the best score (-12.45 kcal/mol) identified within this family is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. It contains four hydroxyl groups, two on each ring. Interestingly, the positioning of these hydroxyls does not allow for the preservation of the hydrogen bond with Gly321. At the opposite end of the molecule, the hydroxyl group in the para position forms a hydrogen bond with the backbone carbonyl of Cys333. Supplementary Fig.\u0026nbsp;9 shows additional compounds belonging to the tolan family. These comprise compounds hydroxylated at different positions and molecules that feature various substituents at one of the rings or condensed rings on one side.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eAlkene to three-membered ring cyclization\u003c/b\u003e: This family consists of compounds in which the central double bond of stilbenes is replaced by a three-membered ring. Interestingly, despite the removal of the central double bond which disrupts the planarity of the molecule, the introduction of a three-membered ring confers rigidity leading to a geometry that closely resembles that of stilbenes. Notably, the two aromatic groups on opposite sides of the ring often lie on the same plane, albeit slightly offset. The compound with the highest docking score (-14.16 kcal/mol) features a cyclopropyl group at the central linker and two condensed rings (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). One of these rings, an indol-2-one, forms an H-bond with Gln383 and Ser365 and is engaged in a π-stacking interaction with His291. The other ring participates in an H-bond with Ser336 and Ser346 and establishes π-stacking interactions with Phe295 and Phe351. Remarkably, this compound, identified through a search based on tapinarof, is closely related to indirubin\u0026mdash;a well-known strong AhR activator with therapeutic potential for psoriatic disease\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. The binding mode of this compound closely resembles that of indirubin, as seen in its experimental structure in complex with the AhR PAS-B\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e (Supplementary Fig.\u0026nbsp;10). Supplementary Fig.\u0026nbsp;11 shows additional compounds belonging to this family. Most of them feature a simple cyclization of the central stilbene double bond (forming either a cyclopropyl or an epoxide group). Interestingly, we also identified some condensed ring systems bearing similarity to well-known AhR ligands, such as BaP. Ligands with aziridine groups were found to bind with less favorable docking scores.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003ePhenyl benzoate derivatives\u003c/b\u003e: This family of compounds was originally inserted into the most promising compounds, as we noted that the substitution of the stilbenes central double bond with an ester group could be a promising modification. However, after refinement of the search through the substructure function, we surprisingly discovered a high number of molecules with docking scores significantly lower than the one obtained by tapinarof, but in which the ester group form an additional condensed cycle (lactonization). Thus, the resulting molecules present three or more condensed aromatic rings. The compound with the highest docking score (-12.95 kcal/mol) presents four condensed rings that establish an extensive π-stacking network with residues Phe295 and Phe351 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Moreover, H-bonds with Ser346, Ser336 and Gly321 anchor the molecule at opposite sides. Supplementary Fig.\u0026nbsp;12 shows additional compounds belonging to this family.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eAlkene to imine/azo\u003c/b\u003e: This family of compounds produces the largest dataset of ligands, with 58,445 compounds retrieved from PubChem. The substitution of one or both carbon atoms in the central double bond of stilbenes (as in N-benzylideneaniline and azobenzene, respectively) provides a flexible scaffold, where the central linker can accommodate an additional fused ring (a feature frequently observed in the phenyl benzoate family as well). Many compounds of this family share a 2-hydroxyquinoxaline ring, with the best compound showing a docking score of -13.84 kcal/mol. This recurrent motif forms H-bonds with Gln383 and Ser365 and engages in π-stacking with His291 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The second ring is stabilized by π-stacking interactions with Phe295 and Phe351. Overall, the two aromatic systems lie almost in the same plane, with an interplanar angle of approximately 20\u0026deg;. Additional compounds from this family are shown in Supplementary Fig.\u0026nbsp;13.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we employed a multistage computational drug-discovery pipeline to explore the chemical space around tapinarof, a known AhR agonist approved for psoriasis treatment. Our docking results on the tapinarof molecule align with the recent experimental data and provide a solid foundation for identifying other compounds capable of activating AhR. By conducting a 3D similarity search in PubChem, we identified 305 compounds, among which we found interesting families. However, these compounds were structurally too similar to tapinarof and were already described in the tapinarof patents. Expanding the search further, we screened 19,943 additional compounds and identified four major promising families with favorable docking scores: tolans, alkene-to-three-membered-ring cyclization, phenyl benzoate derivatives, and alkene-to-imines/azo compounds. The diverse scaffolds identified in this expanded search suggest that non-traditional structural motifs, beyond tapinarof analogues, may offer new therapeutic opportunities for psoriasis and other AhR-related diseases.\u003c/p\u003e \u003cp\u003eInterestingly, we found data supporting further investigation into certain families of compounds. Tolans, for example, are a class of compounds already patented for their use as cosmetics or therapeutics for skin conditions\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Surprisingly, we discovered that the patent is related to the use of tolans as modulator of the sirtuins activity. Sirtuins are a family of enzymes, named after the yeast protein Sir2, which play a crucial role in regulating cellular processes such as aging, stress responses, and metabolism\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. AhR and sirtuins, particularly SIRT1 and SIRT3, exhibit a bidirectional regulatory relationship with implications for skin health and inflammation\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. AhR activation suppresses SIRT1 by reducing NAD\u0026thinsp;+\u0026thinsp;levels, accelerating senescence\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, while SIRT1 enhances AhR-driven processes, including filaggrin expression\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. SIRT1 depletion in keratinocytes has been reported to inhibit both basal and ligand-induced AhR activation, while its presence enhances AhR-driven processes, including AhR/AKT-induced filaggrin expression, which is crucial for skin barrier function\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Moreover, AhR activation inhibits SIRT3 via TiPARP-induced NAD\u0026thinsp;+\u0026thinsp;depletion, increasing oxidative stress through SOD2 acetylation\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. The identification of tolans as AhR modulators suggests they might influence both sirtuins and AhR signaling, key players in psoriasis pathogenesis. Interestingly, tapinarof itself shares structural similarity with resveratrol, piceatannol, and other known sirtuin activators. This raises the possibility that its therapeutic effects may extend beyond AhR activation to include sirtuin modulation. Supporting this, in the work of Smith et al.\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e the molecular profiling experiment evidenced that tapinarof moderately activates SIRT1, while resveratrol\u0026mdash;a well-established SIRT1 activator\u0026mdash;was less potent in the same assay. Given the established crosstalk between AhR and sirtuins, further investigation is warranted to determine whether sirtuin activation contributes to tapinarof efficacy in psoriasis, potentially offering a dual mechanism of action that enhances skin barrier integrity and mitigates inflammation. This dual regulation opens new therapeutic perspectives, where targeting both pathways could help manage inflammation, oxidative stress, and skin barrier function.\u003c/p\u003e \u003cp\u003eOther compounds also emerged as particularly noteworthy. One member of the \u0026ldquo;alkene to three-membered ring cyclization\u0026rdquo; family, Gnetumelin C (Supplementary Fig.\u0026nbsp;11a), is a naturally occurring compound known for its anti-inflammatory, antimicrobial, and antioxidant properties. It has been proposed as a promising ingredient in cosmetic formulations aimed at skin protection and anti-aging applications\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. The top-performing compound within the \u0026ldquo;phenyl benzoate derivatives\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) shares structural similarity with ellagic acid, a naturally occurring heterotetracyclic compound found in various fruits and vegetables. Ellagic acid exhibits antioxidant and anti-proliferative effects and was studied for the topical treatments of melasma\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Additionally, other molecules within the \u0026ldquo;phenyl benzoate derivatives\u0026rdquo; class are particularly interesting, as their unsaturated lactone core represents the structural backbone of coumarin derivatives. These compounds are studied in inflammatory bowel diseases for their ability to activate the AhR/Nrf2 pathways.\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eOverall, our findings underscore the potential of computational drug discovery in identifying novel AhR modulators, broadening the spectrum of candidates for experimental validation. Notably, starting from the pharmacophoric features of tapinarof, our approach identified molecules with potential involvement in other psoriasis-related pathways, as well as compounds structurally similar to established AhR ligands. These results provide a strong foundation for future research and further experimental exploration.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePubChem Search\u003c/h2\u003e \u003cp\u003eTo identify novel AhR modulators, we employed a systematic multi-stage ligand search strategy using the PubChem database\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. PubChem provides several tools for ligand-based searching, including 2D similarity search, 3D similarity search, and substructure search. The multi-stage search strategy is schematized in Supplementary Fig.\u0026nbsp;3 and Supplementary Fig.\u0026nbsp;8.\u003c/p\u003e \u003cp\u003eThe 2D similarity search in PubChem relies on molecular fingerprints, which encode the presence or absence of specific substructural patterns within a molecule. The search algorithm computes a similarity score, typically using the Tanimoto coefficient, to compare the query molecule with the compounds in the database. This approach enables the retrieval of structurally related compounds that share common functional groups and topological features with the query molecule. For our study, we used the PubChem 2D similarity search tool in the second step of the multi-stage search strategy, querying against tapinarof, with the default Tanimoto threshold of 0.9.\u003c/p\u003e \u003cp\u003eUnlike 2D searches, which rely on molecular connectivity, 3D similarity searches evaluate the spatial conformation of molecules. PubChem's 3D search algorithm compares the three-dimensional shape and electrostatic properties of a query molecule against the conformers stored in its database. The scoring function ranks molecules based on shape similarity and pharmacophoric match, providing a set of molecules that may preserve binding modes of the reference ligand. For our study, we used the PubChem 3D similarity search tool in both the first and second steps of the multi-stage search strategy. In the first step, we queried against tapinarof analogs, and in the second step, we expanded the search by querying against 1,3-dichloro-5-(2-phenylethenyl)benzene. The choice of chlorinated trans-stilbene as the target for the 3D similarity search was made because, despite non-hydroxylated trans-stilbenes are known to be good binders of AhR, they were not retrieved in the first 3D similarity search against tapinarof, which predominantly returned molecules with one or more hydrogen donor groups.\u003c/p\u003e \u003cp\u003eThe substructure search tool in PubChem identifies compounds containing a specific molecular core scaffold. This approach is useful for exploring chemical families that share the desired scaffold with the reference compound while allowing for significant variations in the functional groups. To expand our search space, we used the substructure search based on key scaffolds identified in the second step of the multi-stage search. Given that the substructure search may return molecules that differ significantly from the original compound, as long as they contain the searched substructure, we applied filters on size (molecular weight\u0026thinsp;\u0026lt;\u0026thinsp;300 g/mol) and hydrophobicity ( -1\u0026thinsp;\u0026lt;\u0026thinsp;logP\u0026thinsp;\u0026lt;\u0026thinsp;5) to reduce the number of compounds obtained from the search, while remaining close to the queried ligand. The filter on hydrophobicity was not applied to the family of compounds with three-membered ring substitution of the central double bond, as the number of compounds retrieved for this family was already small.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMolecular Docking\u003c/h3\u003e\n\u003cp\u003eThe structure of the human AhR PAS-B domain (PDB ID: 7ZUB\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e) was obtained from the Protein Data Bank. This Cryo-EM structure includes the AhR PAS-B domain complexed with the indirubin ligand and the hsp90 and XAP2 proteins that constitute its cytosolic assembly. For docking calculations, non-AhR proteins were removed, and the resulting structure was preprocessed using Schr\u0026ouml;dinger\u0026rsquo;s Protein Preparation Wizard.\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e Residue protonation states were assigned with PROPKA\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e at pH 7.0.\u003c/p\u003e \u003cp\u003eThe structures of the ligands were downloaded from PubChem in the sdf format and then prepared with the LigPrep utility in the Schr\u0026ouml;dinger 2024-2 suite\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Their protonation states were determined with the Epik Classic tool for pKa prediction included in Maestro\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e, that is based on PROPKA as heuristic pKa calculator.\u003c/p\u003e \u003cp\u003eDocking was performed using Glide XP\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e (extra precision). This method uses a hierarchical series of filters to search for possible locations of the ligand in the binding site and includes a flexible treatment of the ligand. The shape and properties of the protein are represented on a grid by different sets of fields that provide progressively more accurate scoring of the ligand poses. Glide XP performs extensive sampling for ligand positioning through an anchor-and-grow approach and also accounts for explicit waters. The method uses a scoring function (XP GlideScore) that includes force-field-based functions to describe Coulomb and van der Waals contributions to the interaction energy as well as empirically based functions. The receptor grid for the AhR PAS-B domain was centered on the center of mass of the indirubin ligand from the experimental structure. Docking calculations employed the following parameters: a) Retain up to 50,000 poses per ligand during the initial docking phase. b) Use a scoring window of 200 kcal/mol to select initial poses. c) Retain up to 2,000 poses per ligand for energy minimization. d) Apply expanded sampling to increase thoroughness.\u003c/p\u003e\n\u003ch3\u003eLigands Cluster Analysis\u003c/h3\u003e\n\u003cp\u003eTo analyze the results of the virtual screening for the large dataset of ligands obtained in stage 2, we performed a cluster analysis of the ligands that displayed a docking score lower than \u0026minus;\u0026thinsp;9.5 kcal/mol. This threshold was chosen to include only ligands with docking scores within 1 kcal/mol of the score obtained by tapinarof. The subset of ligands meeting this criterion was clustered using the Canvas Similarity and Clustering tool in Maestro. We used a linear fingerprint type with the atom typing scheme 12 (Daylight invariant atom types, where bonds are distinguished by bond order, and cyclic aliphatic structures are distinguished from acyclic aliphatic ones). Similarity was computed using the Tanimoto similarity metric, and results were then clustered using the complete linkage method. The optimal number of clusters was determined based on the Kelley Penalty score. Clusters were analyzed to identify families of ligands that displayed consistently favorable docking scores.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor contributions statement\u003c/h2\u003e\n\u003cp\u003eS.M and L.B. conceived the presented idea, G.S. performed the computations and analyzed the results, S.M supervised the project, L.B. helped supervise the project, G.S. aided in interpreting the results, S.M. drafted the manuscript and designed the figures. All authors discussed the results and reviewed the manuscript.\u003c/p\u003e\n\u003ch2\u003e \u003cb\u003eAdditional information\u003c/b\u003e \u003c/h2\u003e\n\u003cp\u003e \u003cstrong\u003eCompeting interests:\u003c/strong\u003e \u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eS.M and L.B. conceived the presented idea, G.S. performed the computations and analyzed the results, S.M supervised the project, L.B. helped supervise the project, G.S. aided in interpreting the results, S.M. drafted the manuscript and designed the figures. All authors discussed the results and reviewed the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThis research was supported by the National Psoriasis Foundation USA (Discovery Grant - Award ID: 1298983).\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLarigot, L., Juricek, L., Dairou, J. \u0026amp; Coumoul, X. AhR signaling pathways and regulatory functions. \u003cem\u003eBiochim Open\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 1\u0026ndash;9 (2018).\u003c/li\u003e\n\u003cli\u003eEsser, C. \u0026amp; Rannug, A. 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A. \u003cem\u003eet al.\u003c/em\u003e Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes. \u003cem\u003eJ Med Chem\u003c/em\u003e\u003cstrong\u003e49\u003c/strong\u003e, 6177\u0026ndash;96 (2006).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6063129/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6063129/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor involved in the regulation of many patho-physiological processes. Among these, immune system modulation, as well as regulation of skin homeostasis and inflammation, make it a promising target for psoriasis therapy. Tapinarof, an AhR agonist recently approved for psoriasis treatment, exerts its action through antioxidant, anti-inflammatory and barrier-restoring effects. In this study, we employed a computational drug-discovery approach to identify novel AhR modulators with psoriasis therapeutic potential. We performed a multi-step similarity-based screening in PubChem. Application of molecular docking led to the identification of diverse chemical scaffolds with high docking scores and potential AhR activity, some of which belong to chemical classes with known pharmacological relevance. Notably, several identified compounds suggest a possible interplay between AhR signaling and sirtuin modulation, highlighting a previously unexplored avenue in psoriasis therapy. Our findings underscore the potential of computational approaches in accelerating the discovery of novel AhR-targeting agents and provide a foundation for further experimental validation.\u003c/p\u003e","manuscriptTitle":"From Tapinarof to Novel AhR Modulators: Computational Drug Discovery for Psoriasis Therapeutics","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-21 15:47:33","doi":"10.21203/rs.3.rs-6063129/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-03-24T13:21:13+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-19T10:32:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-17T00:22:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"323214328581222918368709880118109906527","date":"2025-03-10T12:13:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"331166699290818574925450309638919318702","date":"2025-03-06T19:32:36+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-06T08:19:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-06T07:55:39+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-02-21T02:55:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-02-19T10:38:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-02-19T10:05:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"70af0899-c199-4a99-bb4b-0cb22f436f82","owner":[],"postedDate":"February 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":44656492,"name":"Biological sciences/Drug discovery/Drug screening/Virtual screening"},{"id":44656493,"name":"Biological sciences/Computational biology and bioinformatics/Virtual drug screening"},{"id":44656494,"name":"Biological sciences/Structural biology/Molecular modelling"},{"id":44656495,"name":"Biological sciences/Drug discovery/Medicinal chemistry/Screening"},{"id":44656496,"name":"Biological sciences/Drug discovery/Medicinal chemistry/Structure based drug design"}],"tags":[],"updatedAt":"2025-06-09T16:00:17+00:00","versionOfRecord":{"articleIdentity":"rs-6063129","link":"https://doi.org/10.1038/s41598-025-03626-z","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-06-06 15:56:56","publishedOnDateReadable":"June 6th, 2025"},"versionCreatedAt":"2025-02-21 15:47:33","video":"","vorDoi":"10.1038/s41598-025-03626-z","vorDoiUrl":"https://doi.org/10.1038/s41598-025-03626-z","workflowStages":[]},"version":"v1","identity":"rs-6063129","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6063129","identity":"rs-6063129","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}