Chemical Research in Toxicology This document is confidential and is proprietary to the American Chemical Society and its authors. Do not copy or disclose without written permission. If you have received this item in error, notify the sender and delete all copies. Identification of potential aryl hydrocarbon receptor ligands by virtual screening of industrial chemicals Journal: Chemical Research in Toxicology Manuscript ID tx-2016-004609 Manuscript Type: Article Date Submitted by the Author: 20-Dec-2016 Complete List of Authors: Larsson, Malin; Umeå University, Department of Chemistry Fraccalvieri, Domenico; Università degli Studi di Milano-Bicocca, 1Dipartimento di Scienze dell’Ambiente e del Territorio Andersson, C. David; Umeå University, Department of Chemistry Bonati, Laura; University of Milano-Bicocca, Department of Earth and Environmental Sciences Linusson, Anna; Umeå University, Department of Chemistry Andersson, Patrik; Umea University, Department of Chemistry ACS Paragon Plus Environment Page 1 of 52 Chemical Research in Toxicology 1 2 3 4 Identification of potential aryl hydrocarbon receptor ligands by virtual 5 6 screening of industrial chemicals 7 8 9 10 Malin Larsson†, Domenico Fraccalvieri‡, C. David Andersson†, Laura Bonati‡, Anna Linusson†, and 11 12 Patrik L. Andersson†* 13 14 15 †Department of Chemistry, Umeå University, SE-901 87 Umeå, Sweden 16 17 18 ‡Department of Earth and Environmental Sciences, University of Milano-Bicocca, Piazza della Scienza 1, 19 20 21 20126 Milano, Italy 22 23 24 *Corresponding author: Tel.: +46-90-786-5266; fax: +46-90-786-7655. Email address: [email protected] 25 26 27 28 29 30 Malin Larsson†: [email protected] 31 32 33 ‡ 34 Domenico Fraccalvieri : [email protected] 35 36 † 37 C. David Andersson : [email protected] 38 39 † 40 Anna Linusson : [email protected] 41 42 ‡ 43 Laura Bonati : [email protected] 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Chemical Research in Toxicology Page 2 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Page 3 of 52 Chemical Research in Toxicology 1 2 3 4 ABSTRACT 5 6 7 We developed a virtual screening procedure to identify potential ligands to the aryl hydrocarbon 8 9 receptor (AhR) among a set of industrial chemicals. AhR is a key target for dioxin-like compounds, 10 11 12 which is related to these compounds potential to induce cancer and a wide range of endocrine and 13 14 immune system related effects. The virtual screening procedure included an initial filtration aiming at 15 16 identifying chemicals with structural similarities to 66 known AhR binders, followed by three 17 18 enrichment methods run in parallel. These include two ligand-based methods (structural fingerprints 19 20 and nearest neighbor analysis) and one structure-based method using an AhR homology model. A set 21 22 of 6,445 commonly used industrial chemicals was processed, and each step identified unique potential 23 24 25 ligands. Seven compounds were identified by all three enrichment methods, and these compounds 26 27 included known activators and suppressors of AhR. Only approximately 0.7% of the studied industrial 28 29 compounds were identified as potential AhR ligands, and of these we suggest that 2-chlorotrityl 30 31 chloride, nisoldipine, 3-p-hydroxyanilino-carbazole, and 3-(2-chloro-4-nitrophenyl)-5-(1,1- 32 33 dimethylethyl)-1,3,4-oxadiazol-2(3H)-one should be addressed in future studies of AhR-mediated 34 35 activities. 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Chemical Research in Toxicology Page 4 of 52 1 2 3 4 1. INTRODUCTION 5 6 7 2,3,7,8-tetrachlorodibenzo-p-dioxin (2378-TCDD) is a persistent organic pollutant that is known to 8 9 induce several toxicological outcomes ranging from acute syndromes such as chloracne in humans to 10 11 12 severe long-term adverse health effects, including immune response-related effects, cancer, and 13 1-5 14 reproductive disorders . AhR plays a central role in the toxicological outcomes of 2378-TCDD, 15 16 which is the most potent known ligand of AhR6. In addition to dioxins and dioxin-like chemicals, AhR 17 18 can bind to and be activated by a number of diverse natural, endogenous, and synthetic compounds6. 19 20 The AhR signaling pathway is also known to cross talk with the estrogen receptor pathway, which 21 22 implies that AhR-activating compounds can result in endocrine disruption7-9. Furthermore, activation 23 24 25 of AhR has also been linked to many immune response processes that are associated with numerous 26 10-16 27 diseases . Thus the identification and regulation of chemicals that induce AhR-related pathways is a 28 29 critical human health and environmental issue. Non-animal testing methods, including in vitro and in 30 31 silico methods, are promoted in the European chemical legislation REACH as ways to identify and 32 33 prioritize chemicals for further testing17. In the United States, the Tox21 and the ToxCast programs 34 35 were initiated to identify hazardous chemicals by using high-throughput screening approaches, 36 37 18-27 38 including large batteries of in vitro assays . In the ToxCast program, 320 chemicals were screened 39 24 40 for five nuclear receptor signaling pathways, including the AhR pathway, and a small percentage of 41 42 these chemicals showed weak AhR-related responses. 43 44 45 Virtual screening is frequently used in medicinal chemistry where in silico methodologies are used to 46 47 48 evaluate large compound libraries for potential associations with a well-defined target, usually a 49 28 50 protein . To evaluate the virtual screening output, the hits are often verified by competitive binding 51 52 assays and/or other in vitro assays29. Virtual screening is based on the structural data of known ligands 53 54 of the studied target (ligand-based screening) and/or characteristics of the target protein (structure- 55 56 based screening)30-38. Ligand-based information includes structural fingerprints and specific chemical 57 58 properties, while structure-based virtual screening is based on interactions between candidate ligands 59 60 and the receptor as evaluated by molecular docking methods37,38. Currently no X-ray crystal structure ACS Paragon Plus Environment Page 5 of 52 Chemical Research in Toxicology 1 2 3 of the AhR ligand binding domain (LBD) is available, and thus homology models have been 4 5 developed to enable detailed studies of ligand interactions36,39-41. The binding free energies of docking 6 7 8 poses obtained from a homology model of the AhR LBD have been shown to correlate well with the 9 10 experimentally derived competitive binding affinities of 14 polychlorinated dibenzo-p-dioxins 11 12 (PCDDs), including 2378-TCDD, indicating that this homology model can be used for virtual 13 14 screening purposes40. 15 16 17 18 The aim of this study was to use virtual screening to identify new potential AhR ligands among a set 19 20 of commonly used industrial chemicals. A virtual screening protocol was developed based on 21 22 structural information from AhR binders that have been shown to induce AhR-related responses and 23 24 on information from a rat AhR homology model40. Ligand similarities were determined by structural 25 26 fingerprints and by nearest neighbor analysis based on 2D-descriptors. Fingerprint-based approaches 27 28 identify similar substances based on their molecular sub-structures, while nearest neighbor analysis 29 30 31 identifies similarities in chemical and structural properties. Protein-ligand interactions were evaluated 32 33 using the binding free energies of the docking poses. When creating the protocol, we used the three 34 35 screening steps in parallel because multiple scoring and data fusion have proven to be more robust 36 37 than, and often outperform, a single virtual screening method34,35,42,43. In the literature search for 38 39 known AhR ligands for the virtual screening protocol, we compiled a database of 214 AhR 40 41 modulators. This study begins with a multivariate analysis of this set of compounds to obtain a broad 42 43 44 overview of the structural and chemical variation of currently known AhR-related compounds. 45 46 47 48 49 50 51 2. MATERIALS AND METHODS 52 53 54 2.1 Datasets 55 56 57 58 We searched the literature for compounds that activate AhR in rat or mouse cell assays, especially 59 60 those whose AhR activity was identified with ethoxyresorufin-O-deethylase (EROD) activity and ACS Paragon Plus Environment Chemical Research in Toxicology Page 6 of 52 1 2 3 dioxin responsive chemically activated luciferase expression (DR-CALUX) assays. These compounds 4 5 are hereafter called “AhR modulators”. The search was performed in SciFinder (2014-09-25) using the 6 7 8 following delimiters: “CALUX dioxin”; “EROD” with refinement “relative potency, not soil, not 9 10 sediment, not contamination, not diet”; and “EROD” with refinement a) “in vitro”, b) “luciferase 11 12 dioxin AhR”, and c) “luciferase AhR”. Reported data from the EROD and CALUX assays showed 13 14 high correlation, and we thus decided to merge these data (Supporting Information). 15 16 17 18 A second database was derived with compounds that bind to AhR, here called the “AhR binders”, that 19 20 were selected based on a reported half maximum inhibition (IC50), inhibition constant (Ki), or 21 22 dissociation constant (Kd) at or below 10 µM as measured in competitive binding assays using labeled 23 24 2378-TCDD in rat or mouse cell systems (Dataset S2).