Tapinarof (GSK2894512) is a naturally derived topical treatment with demonstrated efficacy for patients with psoriasis and atopic dermatitis, although the biologic target and mechanism of action had been unknown. We demonstrate that the anti-inflammatory properties of tapinarof are mediated through activation of the aryl hydrocarbon receptor (AhR). We show that tapinarof binds and activates AhR in multiple cell types, including cells of the target tissue-human skin. In addition, tapinarof moderates proinflammatory cytokine expression in stimulated peripheral blood CD4+ T cells and ex vivo human skin, and impacts barrier gene expression in primary human keratinocytes; both of these processes are likely to be downstream of AhR activation based on current evidence. That the anti-inflammatory properties of tapinarof derive from AhR agonism is conclusively demonstrated using the mouse model of imiquimod-induced psoriasiform skin lesions. Topical treatment of AhR-sufficient mice with tapinarof leads to compound-driven reductions in erythema, epidermal thickening, and tissue cytokine levels. In contrast, tapinarof has no impact on imiquimod-induced skin inflammation in AhR-deficient mice. In summary, these studies identify tapinarof as an AhR agonist and confirm that its efficacy is dependent on AhR.

IntroductionThe SET protein is a potent physiologic inhibitor of protein phosphatase 2A (PP2A) 1 that was isolated from a chromosomal rearrangement at 9q34 in a patient with acute undifferentiated leukemia. 2 The SET protein is overexpressed in chronic myelogenous leukemia (CML) cells, and SET protein levels are further elevated during blast crisis. 3 SET overexpression in CML cells correlates with decreased PP2A activity. 3 This indicates that many of the SET oncogenic activities may be manifest through inhibition of PP2A. PP2A plays a role in many cellular processes, including cell cycle regulation, cell proliferation, apoptosis, development, cytoskeleton dynamics, cell motility, and stem cell self-renewal. 4 In addition, PP2A is a critical tumor suppressor gene that regulates multiple important oncogenic signal transduction pathways. [5][6][7] PP2A inhibition is essential for cell transformation and tumor formation, 8,9 but overexpression of PP2A inhibitory proteins in chronic lymphocytic leukemia (CLL) has not been reported.Of the nearly 84 000 annual cases of leukemia in the Western world, B-cell CLL is the most common, accounting for ϳ 30% of adult leukemia cases. 10 Characterized by accumulation of monoclonal mature B cells, 11 the CLL clinical course is heterogeneous, with some patients experiencing an aggressive course that demands early treatment and others experiencing long survival without disease-related symptoms or ever requiring treatment. 11 Aberrant apoptosis in CLL cells correlates with arrest either in the G 0 or early G 1 phases of the cell cycle. 12,13 This defective apoptosis in CLL cells is partly the result of aberrant signaling through the Akt kinase and the ERK MAPK pathways, in which phosphorylated-Akt is necessary for survival of the leukemia cells. 14,15 The observation of aberrantly activated Akt and downstream pathways in CLL cells also suggests that the normal regulator of these pathways, PP2A, is unable to perform its normal role.We thus sought to determine whether SET is overexpressed in CLL cells relative to normal B cells. We found that SET is significantly overexpressed in CLL cells and related non-Hodgkin lymphoma (NHL) cell line cells. In freshly isolated CLL patient samples, higher cellular levels of the SET correlated with more aggressive disease requiring earlier treatment. Antagonism of SET using shRNA-mediated knockdown or pharmacologic antagonism with novel cell-permeable SET antagonist peptides induced apoptosis, reduced cellular levels of Mcl-1, and caused death of CLL and NHL cells, but normal B cells were scarcely affected by SET antagonism. We also found that pharmacologic SET antagonism in vivo inhibited growth of B-cell NHL tumor xenografts in SCID mice. Methods GeneralAll reagents were from Sigma-Aldrich unless noted otherwise. Anti-SET antibody was from Santa Cruz Biotechnology. Anti-␤-actin, total c-Myc, pS62 c-Myc, and Mcl-1 were from Abcam. All primary antibodies were used at a 1:1000 dilution, except for ␤-actin, which was used at 1:10 000. All secondary ...

The molecular mechanism by which apolipoprotein E (apoE) suppresses inflammatory cytokine and NO production is unknown. Using an affinity purification approach, we found that peptide mimetics of apoE, derived from its receptor binding domain residues 130–150, bound to the SET protein, which is a potent physiological inhibitor of protein phosphatase 2A (PP2A). Both holo-apoE protein and apoE-mimetic peptides bound to the C-terminal region of SET, which is then associated with an increase in PP2A-mediated phosphatase activity. As physiological substrates for PP2A, the LPS-induced phosphorylation status of signaling MAPK and Akt kinase is reduced following treatment with apoE-mimetic peptides. On the basis of our previous report, in which apoE-mimetic peptides reduced I-κB kinase and NF-κB activation, we also demonstrate a mechanism for reduced production of inducible NO synthase protein and its NO product. These data provide evidence for a novel molecular mechanism by which apoE and apoE-mimetic peptides antagonize SET, thereby enhancing endogenous PP2A phosphatase activity, which reduces levels of phosphorylated kinases, signaling, and inflammatory response.

BackgroundPsoriasis is a chronic inflammatory skin disorder involving marked immunological changes. IL-17-targeting biologics have been successful in reducing the disease burden of psoriasis patients with moderate-to-severe disease. Unfortunately, the stratum corneum prevents penetration of large molecule weight proteins, including monoclonal antibodies. Thus, for the majority of psoriasis patients ineligible for systemic treatments, a small molecule targeting RORγt, the master regulator of IL-17 family cytokines, may represent an alternative topical medicine with biologic-like efficacy.Methods and FindingsThe preclinical studies described in this manuscript bridge the gap from bench to bedside to provide the scientific foundation for a compound entering clinical trials for patients with mild to moderate psoriasis. In addition to several ex vivo reporter assays, primary T cell cultures, and the imiquimod mouse model, we demonstrate efficacy in a newly developed human ex vivo skin assay, where Th17-skewed cytokine expression is induced from skin-resident immune cells. Importantly, the skin barrier remains intact allowing for the demonstration of topical drug delivery. With the development of this novel assay, we demonstrate potent compound activity in the target tissue: human skin. Finally, target engagement by this small molecule was confirmed in ex vivo lesional psoriatic skin.ConclusionsOur work describes a progressive series of assays to demonstrate the potential clinical value of a novel RORγ inverse agonist small molecule with high potency and selectivity, which will enter clinical trials in late 2015 for psoriasis patients.