GSK2981278 targets RORγ (RORC protein, human, Nuclear Receptor Subfamily 1, Group F, Member 3), acting as a selective inverse agonist of RORγ [1]

GSK2981278 significantly and effectively inhibited IL-17A and IL-22 proteins in a concentration-dependent manner (IC50=3.2 nM) during 5 days of culture under Th17 tilt conditions [1]. GSK2981278 (0.3, 1, 3, 10, 30). effectively and locally suppresses IL-17 and IL-22 levels (at doses of 100, 300, and 1000 pM for five days). IL-17A polypeptide is nearly completely inhibited when incubated with ≥ 3 nM GSK2981278 [1].

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1. GSK2981278 potently inhibited RORE-dependent activation in a Luciferase-based reporter (pGL4-27-5xRORE) CHO Tet-on cell line in a concentration-dependent manner (percent inhibition calculated relative to DMSO vehicle), with no significant effect on cell viability measured by cAMP-based assay [1]
2. GSK2981278 inhibited activation of the human il17 promoter in compound-treated Jurkat cells co-transfected with control or il17 promoter reporter plasmid (significant inhibition, p≤0.01 by Student’s t test) [1]
3. Mammalian mono-hybrid and two-hybrid assays showed that GSK2981278 suppressed RORγ-mediated transcriptional activity in Jurkat cells co-transfected with control or reporter plasmids (significant inhibition, p≤0.01 by Student’s t test) [1]
4. In pCMV10-3xFlag-RORγ-transfected Jurkat cells stimulated for 24 hours, GSK2981278 significantly reduced il17 mRNA levels (qRT-PCR, p≤0.01 by Student’s t test) [1]
5. ChIP-qPCR in pCMV10-3xFlag-RORγ-transfected Jurkat cells confirmed that GSK2981278 inhibited RORγ binding to the il17 promoter region (significant inhibition, p≤0.01 by Student’s t test) [1]
6. In human skin explant models (Skin Resident Immune Cell Activation, sRICA assay), pre-treatment with 10 μM GSK2981278 for 1 day prior to 24–48 hours of Th17 stimulation significantly reduced Th17-skewed pro-inflammatory cytokine transcript levels (qRT-PCR, p≤0.05/p≤0.01/p≤0.001 by Student’s t test) while maintaining tissue integrity (H&E staining) [1]
7. In ex vivo human skin cultured in Franz Cell chambers, topical application of GSK2981278 at time zero (followed by Th17 polarization at 24 hours) significantly reduced il17a and il17f gene expression in skin sections harvested after 24 hours of stimulation (48 hours of compound treatment), measured as percent maximum expression relative to vehicle-treated samples (significant inhibition, p≤0.05 by Student’s t test) [1]
8. In ex vivo psoriatic skin biopsies (3-4mm punch biopsy) cultured in Cornification media with 10 μM GSK2981278 for 12–14 hours (0.2% DMSO as control), proinflammatory cytokine levels were significantly reduced (percent inhibition relative to DMSO control, p≤0.01 by Paired t-test) [1]

GSK2981278 (1% soft; topical; 3 days) decreases skin repair and redness, as shown by a 23% decrease in skin thickness. GSK2981278 reliance in a model of raccoons [1].

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1. In the imiquimod (IMQ)-induced mouse model of psoriasis, topical treatment with 1% GSK2981278 in ointment significantly reduced epidermal thickening (mean epidermal thickness measured across 6–9 mice per group, p≤0.05 by Student’s t test) and skin inflammation (H&E staining of back skin at day +9 of IMQ treatment) compared to placebo ointment [1]
2. Topical application of 1% or 0.1% GSK2981278 in a 60:40 ethanol:water solution to IMQ-induced psoriasis mice significantly reduced local pro-inflammatory cytokine expression in skin tissues at day +3 and day +9 of IMQ treatment (mean ± SEM gene expression across 6–9 mice per group, p≤0.05/p≤0.01 by Student’s t test) [1]

1. RORγ-mediated RORE reporter assay: CHO Tet-on cells stably transfected with pGL4-27-5xRORE (RORE-dependent Luciferase reporter) were treated with different concentrations of GSK2981278 (DMSO as vehicle control); Luciferase activity was measured to calculate percent inhibition of RORE-dependent activation, and cAMP-based cell viability assay was performed to exclude non-specific cytotoxic effects [1]
2. IL-17 promoter activation assay: Jurkat cells were co-transfected with control plasmid or human il17 promoter reporter plasmid, then treated with GSK2981278 (DMSO as control); Luciferase activity was measured to evaluate inhibition of il17 promoter activation, with statistical significance determined by Student’s t test [1]
3. RORγ transcriptional activity assay (mammalian mono-hybrid/two-hybrid): Jurkat cells were co-transfected with control plasmids or RORγ-specific reporter plasmids (mono-hybrid/two-hybrid system), then treated with GSK2981278 or vehicle; reporter gene activity was measured to assess RORγ transcriptional activity inhibition [1]
4. ChIP-qPCR assay for RORγ binding to IL-17 promoter: pCMV10-3xFlag-RORγ-transfected Jurkat cells were treated with GSK2981278 or vehicle, then cross-linked and immunoprecipitated with anti-Flag antibody; qPCR was performed to quantify RORγ binding to the il17 promoter region, with statistical analysis by Student’s t test [1]

1. Jurkat cell IL-17 mRNA expression assay: Jurkat cells were transfected with pCMV10-3xFlag-RORγ plasmid, stimulated for 24 hours with/without GSK2981278; total RNA was extracted, and il17 mRNA levels were quantified by qRT-PCR (DMSO as control), with statistical significance determined by Student’s t test [1]
2. Human skin explant sRICA assay: Human skin explants were cultured for ≥4 days to maintain tissue integrity (H&E staining verification); explants were pre-treated with 10 μM GSK2981278 or DMSO for 1 day, then stimulated under Th17-polarizing conditions for 24–48 hours; total RNA was extracted, and pro-inflammatory cytokine transcript levels were measured by qRT-PCR (percent maximum stimulation calculated from 3 independent experiments, Student’s t test for significance) [1]
3. Ex vivo human skin Franz Cell assay: Human skin samples were placed in Franz Cell chambers and cultured for 48 hours; GSK2981278 was topically applied to the dry skin surface at time zero, followed by Th17 polarization of skin-resident immune cells at 24 hours; skin sections were harvested at 48 hours, total RNA was extracted, and il17a/il17f gene expression was quantified by qRT-PCR (percent maximum expression relative to vehicle control, Student’s t test for significance) [1]
4. Psoriatic skin biopsy assay: Psoriatic skin biopsies (3-4mm punch) were shipped overnight in Unisol buffer, then cultured in Cornification media with 10 μM GSK2981278 or 0.2% DMSO for 12–14 hours (no external stimulation); cytokine levels were measured, and percent inhibition relative to DMSO control was calculated (Paired t-test for significance) [1]

Animal/Disease Models: BALB/c JByRj female mice (8 weeks old at study entry; imiquimod (IMQ) mouse model) [1]
Doses: 1%
Route of Administration: Ointment; Topical; Three Day
Experimental Results: Skin Redness and scales are diminished, hyperplasia is diminished, and epidermal thickness is diminished by 23%.

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1. Imiquimod-induced psoriasis mouse model (ointment formulation): BALB/c mice were treated topically with placebo ointment or 1% GSK2981278 in ointment, combined with IMQ (psoriasis induction) or petrolatum (vehicle control); back skin was treated daily for 9 days (day +9 of IMQ treatment); at study endpoint, back skin was imaged, H&E stained, and mean epidermal thickness was measured across 6–9 mice per group [1]
2. Imiquimod-induced psoriasis mouse model (ethanol:water solution): BALB/c mice with IMQ-induced psoriasis were treated topically with 1% or 0.1% GSK2981278 in a 60:40 ethanol:water solution (placebo solution as control); skin tissues were collected at day +3 and day +9 of IMQ treatment (6–9 mice per group); pro-inflammatory cytokine gene expression was measured by qRT-PCR (mean ± SEM, Student’s t test for significance) [1]

[1]. Development of a Topical Treatment for Psoriasis Targeting RORγ: From Bench to Skin. PLoS One. 2016 Feb 12;11(2):e0147979.

GSK-2981278 is being studied in the clinical trial NCT03004846 (a study of GSK2981278 ointment in patients with plaque psoriasis).

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1. GSK2981278 is a novel, highly effective and selective RORγ reverse agonist small molecule developed for the topical treatment of mild to moderate psoriasis[1]
2. Psoriasis is a chronic inflammatory skin disease driven by IL-17 family cytokines (regulated by RORγt, the major regulator of Th17 cells); IL-17-targeting biologics are highly effective but cannot penetrate the stratum corneum, so small molecule RORγ inhibitors have become a promising alternative for topical treatment[1]
3. GSK2981278 has shown target binding ability in isolated psoriatic lesions and exhibits potent activity in human skin (target tissue) while maintaining the integrity of the skin barrier[1]
4. GSK2981278 entered clinical trials for mild to moderate psoriasis at the end of 2015, and preclinical data support its efficacy similar to biologics when administered topically[1]

C25H35NO5S

461.614106416702

461.223

C, 65.05; H, 7.64; N, 3.03; O, 17.33; S, 6.95

1474110-21-8

89875987

White to off-white solid powder

1.2±0.1 g/cm3

622.6±65.0 °C at 760 mmHg

330.3±34.3 °C

0.0±1.9 mmHg at 25°C

1.564

4

1

6

10

32

635

0

S(C1C=CC(=C(CO)C=1)OCC1CCOCC1)(N(C1C=CC(CC)=CC=1)CC(C)C)(=O)=O

LZLBRISQTJVZNP-UHFFFAOYSA-N

InChI=1S/C25H35NO5S/c1-4-20-5-7-23(8-6-20)26(16-19(2)3)32(28,29)24-9-10-25(22(15-24)17-27)31-18-21-11-13-30-14-12-21/h5-10,15,19,21,27H,4,11-14,16-18H2,1-3H3

N-(4-ethylphenyl)-3-(hydroxymethyl)-N-(2-methylpropyl)-4-(oxan-4-ylmethoxy)benzenesulfonamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.42 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.42 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.42 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)