Development of an Aryl Hydrocarbon Receptor Antagonist Using the Proteolysis-Targeting Chimeric Molecules Approach: A Potential Tool for Chemoprevention Dinesh Puppala, Hyosung Lee, Kyung Bo Kim, and Hollie I. Swanson Department of Molecular and Biomedical Pharmacology (D.P., H.I.S.), and Department of Pharmaceutical Sciences (H.L. and K.B.K.) University of Kentucky, Lexington, Kentucky Received August 10, 2007; accepted January 3, 2008 ABSTRACT Activation of the aryl hydrocarbon receptor (AHR) by agonists and environmental contaminants like dioxin (2,3,7,8-tetrachloro- dibenzo-p-dioxin) leads to many adverse biological effects, including tumor promotion. With this in mind, we propose that agents that block the AHR pathway may be therapeutically beneficial, particularly by exhibiting chemopreventive activ- ities. In our current research, we have focused on the devel- opment of an AHR antagonist using a chemical genetic ap- proach called PROTACS (PROteolysis-TArgeting Chimeric moleculeS). PROTACS is a novel approach of tagging small recognition sequences of a specific E3 ubiquitin ligase com- plex to a known ligand for the receptor of interest (AHR) for targeting its degradation. Here, we present the design and initial characterization of AHR targeting PROTACS (Apigenin- Protac) designed to degrade and inhibit the AHR in epithelial cells. Our results demonstrate the “proof of concept” of this approach in effectively blocking AHR activity in cultured cells. Exposures to environmental factors are believed to play a substantial role in the development of many human cancers (Boffetta and Nyberg, 2003; Boffetta, 2004; Poirier, 2004; Luch, 2005). For example, in a Swedish study that encom- passed 9.6 million individuals, it was reported that environ- mental factors account for 69% of colon cancers, 79% of lung cancers, and 78% of kidney cancers (Czene et al., 2002). Although the specific causative agents are difficult to iden- tify, one class of human carcinogens that is believed to be important because of its high prevalence in the environment and relatively well-characterized mode of action in animal models is that of the aromatic hydrocarbons (Luch, 2005). Members of this group include benzo[a]pyrene, polychlori- nated biphenyls, and 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD), which are contaminants present in cigarette smoke and other environmental sources. The carcinogenic actions of many polycyclic aromatic hydrocarbons typically occur after their binding to and activation of the aryl hydrocarbon re- ceptor (AHR). Although the AHR is typically believed to be involved largely in the genotoxic actions of environmental carcinogens, emerging evidence indicates a role of the AHR in tumor promotion and progression (Luch, 2005). At the cellular level, the AHR is present in the cytoplasm as a component of an AHR chaperone complex in association with heat shock protein-90, XAP2/ARA9, and p23 (Kewley et al., 2004). Upon activation by its agonists, the AHR translo- cates into the nucleus, where it dissociates from its chaper- one complex and binds with its dimerization partner aryl hydrocarbon receptor nuclear translocator (ARNT). The AHR-ARNT dimer then interacts with its DNA recognition sites, dioxin response elements (DREs), and subsequently regulates a battery of AHR target genes such as CYP1A1 and CYP1B1. Although CYP1A1 and CYP1B1 are perhaps the best-characterized AHR target genes and are typically con- sidered to be biomarkers of the AHR pathway, it has not yet been established whether up-regulation of CYP1A1 and CYP1B1 is required for all of the carcinogenic effects elicited by AHR agonists. The promise associated with using the AHR as a target for effective chemopreventive approaches has been demonstrated by the use of AHR antagonists like 3'-methoxy-4'-nitroflavone This work was supported by National Institutes of Health grants ES11295, ES08088, and ES80422. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.107.040840. ABBREVIATIONS: AHR, aryl hydrocarbon receptor; ARNT, aryl hydrocarbon receptor nuclear translocator; MNF, 3'- methoxy-4'-nitroflavone; NHK, normal primary human keratinocyte; PROTACS, Proteolysis-targeting chimeric molecules; and TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; DRE, dioxin response element; DMSO, dimethyl sulfoxide; RT, reverse transcriptase; PCR, polymerase chain reaction; ANOVA, analysis of variance; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; EMSA, electrophoretic mobility shift assay. 0026-895X/08/7304-1064 –1071$20.00 MOLECULAR PHARMACOLOGY Vol. 73, No. 4 Copyright © 2008 The American Society for Pharmacology and Experimental Therapeutics 40840/3315565 Mol Pharmacol 73:1064–1071, 2008 Printed in U.S.A. 1064 at ASPET Journals on August 9, 2017 molpharm.aspetjournals.org Downloaded from