Fig 1.
GSK2981278 is a strong RORγ-selective inverse agonist that inhibits activation of the il17 promoter.
(A) RORE-dependent activation was examined using a Luciferase-based reporter (pGL4-27-5xRORE) CHO Tet-on cell line. Percent inhibition was calculated relative to vehicle (DMSO). cAMP-based cell viability was measured. (B) Activation of the human il17 promoter in compound-treated Jurkat cells co-transfected with control or reporter plasmid. D, indicates DMSO. (C-D) Mammalian mono-hybrid (C) or two-hybrid (D) analysis was performed following co-transfection with control or reporter plasmids +/- compound. (E) pCMV10-3xFlag-RORγ-transfected Jurkat cells were stimulated for 24 hrs and il17 mRNA levels determined by qRT-PCR. (F) ChIP-qPCR was performed in pCMV10-3xFlag-RORγ-transfected Jurkat cells. All experiments were independently performed at least twice. Significant inhibition was determined by Student’s t test. (* p≤0.01).
Fig 2.
GSK2981278 attenuates inflammation in a mouse model of psoriasis.
(A) Mice were treated topically with placebo or 1% GSK2981278 (1278) in ointment, and with imiquimod (IMQ) or petrolatum (vehicle). At study’s end (day +9 of IMQ treatment), back skin was imaged and stained (H&E). (B) Mean epidermal thickness is shown across 6–9 mice per treatment group. (C-E) Changes to local cytokine expression was determined following topical application of 1% or 0.1% compound in a simple ethanolic solution (60:40 ethanol:water). (C) Description of study groups for panels D-E. Skin cytokine levels on day +3 (D) or day +9 (E). Data reflect the mean ± SEM gene expression level across 6–9 mice per treatment group. Significant inhibition was determined by Student’s t test. (*p≤0.05;**p≤0.01).
Fig 3.
Skin Resident Immune Cell Activation (sRICA) leads to pro-inflammatory cytokine responses that are reduced by GSK2981278.
(A) Skin explants were cultured ≥4 days under the indicated conditions. Explants were analyzed for tissue integrity by H&E. (B) Samples were pre-treated with 10 μM compound (closed bar) or vehicle (DMSO; open bars–set to 100%) for 1 day prior to 24–48 hrs of Th17 stimulation. Relative transcript levels were determined by qRT-PCR. (C) Samples were treated as in B, then analyzed daily for tissue integrity by H&E. Images are representative at least 3 independent experiments. (D) Samples were treated as in B. Graphs show the mean percent maximum stimulation of 3 independent experiments. Significant inhibition was determined by Student’s t test. (*p≤0.05; **p≤0.01; ***p≤0.001).
Fig 4.
Suppression of Th17-type cytokine production following topical application.
Ex vivo human skin was cultured in Franz Cell chambers for a total of 48 hours. GSK2981278 was applied to the dry surface of the skin at time zero followed 24 hrs later by activation of skin resident immune cells under Th17 polarizing conditions. The experimental schema is shown in panel A. Skin sections were harvested after 24 hrs of stimulation (48 hrs of compound treatment) and analyzed for relative gene expression of il17a (B) or il17f (C). Data are shown as the percent maximum expression of each cytokine as compared to Th17-stimulated samples treated topically with vehicle only.
Fig 5.
Treatment of psoriatic tissue with the RORγ inverse agonist GSK2981278 reduces proinflammatory cytokine levels.
Three psoriatic skin biopsies were obtained via 3-4mm punch biopsy and placed in Unisol buffer for overnight shipment. Upon arrival, biopsy sections were placed in Cornification media without stimulation for 12–14 hours with either 0.2% DMSO or 10 μM compound. The percent inhibition of each biomarker compared to culture with DMSO alone is shown. Each point represents an individual patient sample. The percent maximum expression ± SEM for each analyte assayed of each tissue was calculated relative to time zero. Significant inhibition was determined by Paired t-test. (**p≤0.01).