Tapinarof Is a Natural AhR Agonist that Resolves Skin Inflammation in Mice and Humans  Susan H. Smith, Channa Jayawickreme, David J. Rickard, Edwige Nicodeme, Thi Bui, Cathy Simmons, Christine M. Coquery, Jessica Neil, William M. Pryor, David Mayhew, Deepak K. Rajpal, Katrina Creech, Sylvia Furst, James Lee, Dalei Wu, Fraydoon Rastinejad, Timothy M. Willson, Fabrice Viviani, David C. Morris, John T. Moore, Javier Cote-Sierra  Journal of Investigative Dermatology  Volume 137, Issue 10, Pages 2110-2119 (October 2017) DOI: 10.1016/j.jid.2017.05.004 Copyright © 2017 The Authors Terms and Conditions

Figure 1 Structural similarities and unique activity profiles of tapinarof and resveratrol. (a) Chemical structure of tapinarof (GSK2894512), (b) chemical structure of resveratrol, and (c) activity profile of tapinarof (red) and resveratrol (black) across a selection of assays (defined in Supplementary Table S1). No activity and that below the level of quantification are arbitrarily set to a value of 2 (10 mM). Journal of Investigative Dermatology 2017 137, 2110-2119DOI: (10.1016/j.jid.2017.05.004) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Tapinarof activates the AhR pathway through direct binding. (a) BioMap analysis across multiple doses. The shaded gray area represents normal variation. Analytes outside normal variation are annotated. (b) CYP1A1 mRNA after 3 days (CD4+ T cells) or overnight culture (human skin) with titrating concentrations of tapinarof. Samples normalized to GAPDH are shown relative to DMSO control as the average ± SEM from three donors tested independently. (c) AhR nuclear translocation was determined in HaCaT cells after 30 minutes with tapinarof. Data are representative of four experiments with similar results. (d) Intrinsic fluorescence signals (arbitrary scale) of 100 nM tapinarof (closed squares) or 100 nM resveratrol (closed circles; used as a structurally related polyphenol control) were measured after overnight incubation with human (left) or mouse (right) AHR-ARNT heterodimers. AhR, aryl hydrocarbon receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SEM, standard error of the mean. Journal of Investigative Dermatology 2017 137, 2110-2119DOI: (10.1016/j.jid.2017.05.004) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Tapinarof induces barrier gene expression in keratinocytes. (a) Confluent primary human keratinocytes were treated 72 hours with DMSO or tapinarof (0.1 and 1 μM). CYP1A1 expression relative to DMSO-treated cells after normalizing to β-actin. (b) Keratinocyte viability. Data show mean ± SEM of 10 replicates. (c) mRNA expression levels of genes related to keratinocyte differentiation (KRT1, DSC1, LOR, FLG, HRNR, and IVL), in confluent primary keratinocytes after 72 hours of treatment. Data normalized to β-actin are shown relative to DMSO-treated. Data in (a) and (c) are combined from two donors in four independent experiments. Statistical significance was determined by one-way analysis of variance using a Tukey posttest on ΔCT values and noted where significant for both test concentrations (**P < 0.01, ***P < 0.001). SEM, standard error of the mean. Journal of Investigative Dermatology 2017 137, 2110-2119DOI: (10.1016/j.jid.2017.05.004) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Treatment with tapinarof leads to preclinical improvement in a mouse model of psoriasis. (a–d) Mice were pretreated with tapinarof or vehicle control for 3 days. Imiquimod or vanicream control was introduced day 0 and mice followed ≤9 days. (a) Representative image (day 9) is shown for each condition. (b) Clinical score monitored daily according to the scale shown in Supplementary Table S5. (c) Epidermal thickness at study termination. (d) mRNA transcript levels from isolated back skin normalized to β-actin and shown relative to imiquimod-treated animals. (b–d) Data are combined from 9 to 10 mice/treatment and shown as the mean ± standard error. Significant differences as determined by one-way analysis of variance using a Tukey posttest are noted (*P < 0.05, ***P < 0.001). IMQ, imiquimod. Journal of Investigative Dermatology 2017 137, 2110-2119DOI: (10.1016/j.jid.2017.05.004) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Tapinarof does not protect AhR knockout mice from IMQ-induced psoriasiform inflammation. AhR-sufficient (+/−) and -deficient (−/−) mice were treated with tapinarof (1%), FICZ (0.01%, the limit of solubility) or vehicle control, and IMQ or vanicream control similar to Figure 4. (a) Clinical scores for AhR-sufficient (left plot) and AhR-deficient (right plot) animals are reported according to the scale in Supplementary Table S6 online. (b, c) Changes in cytokine transcript levels were measured at study completion from isolated back skin. Data were normalized to β-actin and percent affected was determined by setting expression in imiquimod-treated animals to 100%. Data shown are combined results of ≥25 mice per treatment group over two independent studies. Significant differences by the one-way analysis of variance and Tukey posttest are noted (***P < 0.001). AhR, aryl hydrocarbon receptor; FICZ, 6-formylindolo(3,2-b)carbazole; IMQ, imiquimod. Journal of Investigative Dermatology 2017 137, 2110-2119DOI: (10.1016/j.jid.2017.05.004) Copyright © 2017 The Authors Terms and Conditions