GNF351 (500 nM, 48 hours) treatment of proliferating monolayers of human ex vivo cell cultures considerably lowers the total number of Ki67-positive cells and cell number [1].

GNF351 (500 nM, 48 hours) treatment of proliferating monolayers of human ex vivo cell cultures considerably lowers the total number of Ki67-positive cells and cell number [1].

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GNF351 (500 nM) suppressed the expression of both early (e.g., Krt1, Pou2f3) and late (e.g., Ivl, Lor, Dsc1) epidermal differentiation genes in primary mouse keratinocytes cultured under both basal (0.05 mM Ca²⁺) and differentiation-inducing (0.12 mM Ca²⁺) conditions. [1]
GNF351 (500 nM) completely blocked the induction of the classic AHR target gene Cyp1a1 in differentiating mouse keratinocytes. [1]
GNF351 (500 nM) significantly downregulated the expression of the cytokine genes Il33 and Il36g in primary mouse keratinocytes. [1]
GNF351 (500 nM) blocked the induction of keratin 10 and loricrin protein expression in differentiating primary mouse keratinocytes, as confirmed by immunoblot analysis. [1]
In primary human keratinocytes induced to differentiate by growth factor depletion, treatment with GNF351 (500 nM) reduced the expression of differentiation markers CYP1A1, FLG (filaggrin), HRNR (hornerin), and LOR (loricrin). [1]
GNF351 (500 nM) decreased the protein levels of pro-filaggrin, involucrin, and loricrin in differentiating human primary keratinocytes, as shown by immunoblot analysis. [1]
GNF351 (500 nM) treatment of proliferating monolayer cultures of human primary keratinocytes for 48 hours significantly reduced the percentage of Ki67-positive cells and the total cell count, indicating suppression of proliferation. [1]
In primary mouse keratinocytes induced to differentiate, treatment with GNF351 (200 nM) for 24 hours increased the levels of AHR protein retained in nuclear extracts, while it also increased cytoplasmic AHR levels. [1]
In proliferating primary mouse keratinocytes, treatment with GNF351 (200 nM) caused a slower and sustained increase in nuclear AHR levels over 24 hours, unlike rapid agonists such as TCDD. [1]

Cell Proliferation Assay[1]
Cell Types: Human Primary keratin cells
Tested Concentrations: 500 nM
Incubation Duration: 48 hrs (hours)
Experimental Results: Significant percentage of Ki67-positive cells and cell number after 48 hrs (hours) of treatment of proliferating human keratinocyte monolayer cultures reduce.

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Mouse Keratinocyte Differentiation Gene Expression: Primary mouse keratinocytes isolated from newborn mice were cultured in low-calcium (0.05 mM CaCl₂) medium for proliferation. To induce differentiation, the medium was switched to high-calcium (0.12 mM CaCl₂) medium. Cells were treated with GNF351 (500 nM) or vehicle control. After specified times, total RNA was isolated and quantitative RT-PCR (qPCR) was performed to measure the expression of differentiation genes (e.g., Krt1, Lor, Ivl, Dsc1, Pou2f3, Cyp1a1) and cytokine genes (e.g., Il33, Il36g). Gene expression was normalized to Gapdh. [1]
Human Keratinocyte Differentiation Gene Expression: Human primary keratinocytes were isolated from abdominal skin and cultured in keratinocyte growth medium (KGM). Differentiation was induced by growth factor depletion. Cells were treated with GNF351 (500 nM) or vehicle control. RNA was extracted, and qPCR was performed to assess the expression of human differentiation markers (CYP1A1, FLG, HRNR, LOR), normalized to RPLP0. [1]
Western Blot Analysis for Protein Expression: Mouse or human keratinocytes were treated with GNF351 under proliferation or differentiation conditions. Cells were lysed, and proteins were extracted. Proteins were separated by electrophoresis, transferred to membranes, and probed with specific primary antibodies (e.g., against keratin 10, loricrin, involucrin, pro-filaggrin, AHR). Detection was performed using appropriate secondary antibodies and an enhanced chemiluminescence method. [1]
Cell Proliferation Assay: Human primary keratinocytes in monolayer culture during the proliferation phase were treated with GNF351 (500 nM) for 48 hours. Cells were fixed, permeabilized, and immunostained for the proliferation marker Ki-67. Nuclei were counterstained with DAPI. The percentage of Ki-67 positive cells was quantified. Total cell counts were also performed. [1]
Subcellular Fractionation and AHR Localization: Primary mouse keratinocytes were treated with GNF351 (200 nM) under proliferation or differentiation conditions. At specified time points, cytoplasmic and nuclear protein fractions were isolated separately. The levels of AHR in each fraction were analyzed by Western blotting, using tubulin and lamin A/C as markers for cytoplasmic and nuclear fractions, respectively. [1]

The study indicated that treatment of mouse or human keratinocyte cultures with AHR ligands (including GNF351) at the concentrations used (e.g., 500 nM) showed extremely low toxicity. However, specific toxicity data (e.g., LD50, organ toxicity) were not provided. [1]

[1]. Genetic and pharmacological analysis identifies a physiological role for the AHR in epidermal differentiation. J Invest Dermatol. 2015 May;135(5):1320-1328.

GNF351 is described as a complete antagonist of both dioxin-responsive element (DRE)-dependent and DRE-independent aryl hydrocarbon receptor (AHR) function. It blocks agonist-induced AHR target gene expression, but does not possess agonist activity itself. [1]
Its mechanism of action involves direct binding to the AHR ligand-binding pocket, thereby preventing agonist-induced activation and subsequent gene transcription. [1]
This study shows that pharmacological inhibition of the AHR signaling pathway using antagonists such as GNF351 impairs normal epidermal differentiation and stratification in a human skin equivalent model, suggesting that endogenous AHR activity is crucial for maintaining skin homeostasis. [1]
These findings suggest that GNF351 and similar AHR antagonists or selective modulators may be potential drugs for treating skin diseases characterized by dysdifferentiation or inflammation. [1]

C24H25N7

411.502203702927

411.217

1227634-69-6

46216378

Off-white to light yellow solid powder

4.1

2

5

6

31

586

0

N1(C=NC2=C(N=C(C3C=NC=C(C)C=3)N=C12)NCCC1=CNC2C=CC=CC1=2)C(C)C

WWERDIHAUJLVQP-UHFFFAOYSA-N

InChI=1S/C24H25N7/c1-14(2)31-20(9-8-16-13-27-19-7-5-4-6-18(16)19)28-21-22(25)29-23(30-24(21)31)17-10-15(3)11-26-12-17/h4-7,10-14,27H,8-9H2,1-3H3,(H2,25,29,30)

8-(2-(1H-indol-3-yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine

GNF-351; GNF351 GNF 351.

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)

DMSO : ≥ 125 mg/mL (~303.77 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.05 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 20.8 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: ≥ 2.08 mg/mL (5.05 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 20.8 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.)