Fig 1.
T-3833261 is a potent ATP competitive PRS inhibitor.
(A) Molecular model constructed by available PRS crystal structures bearing adenosine and proline (PDB: 4K87) and Halofuginone (PDB: 4K88), (B) Binding mode of T-3833261 in the ATP binding site, (C) Chemical structure, (D) Biochemical activity of compound in PRS inhibition measured by an ATP/PPi exchange method. Results performed in duplicate are shown as the mean ± SD. The inhibitory activities are expressed as the percentage of vehicle-treated control.
Fig 2.
Effect of T-3833261 or Halofuginone on α-SMA and pro-COL1A1 protein content in TGF-β-treated human skin fibroblast.
Skin fibroblasts were pre-treated with samples (1–300 nM) for 0.5 h, followed by stimulation with TGF-β (1 ng/mL). After incubation for 24 h, α-SMA (A) and pro-COL1A1 (B) protein levels were measured by ELISA. The expression levels are expressed as the percentage of vehicle-treated control. Values are mean ± SD (n = 4). #p<0.05 compared to vehicle-treated control, *p<0.05 compared to TGF-β-treated control. The experiment was repeated by using other fibroblast lots and similar results were obtained.
Fig 3.
Effect of T-3833261 or Halofuginone on Smad3 protein expression in human skin fibroblasts.
Representative western blots show that Halofuginone or T-3833261 reduces Smad3 and phosphorylated Smad3 (p-Smad3) protein expressions significantly. β-actin shown as simultaneous loading controls. The data of this experiments were repeated three times.
Fig 4.
Effects of T-3833261 or Halofuginone on mRNA expression of fibrosis-related genes.
Skin fibroblasts were pre-treated with samples (1–300 nM), followed by stimulation with TGF-β (1 ng/mL). After incubation for 24 h, α-SMA, COL1A1 (A), DDIT3, FGF2 and SMURF2 (B) mRNA levels were measured by real-time quantitative RT-PCR. Gene expression levels (normalized to GAPDH) are expressed as the fold change of vehicle-treated control. Values are mean ± SD (n = 6). #p<0.05 compared to vehicle-treated control, *p<0.05 compared to TGF-β-treated control. The experiment was repeated by using other fibroblast lots and similar results were obtained.
Fig 5.
Fibrotic genes expression of ear and dorsal skin in a mouse by TGF-β injection.
TGF-β (500 ng/20 μL) or vehicle was intradermally injected 3 or 5 times in the ear (A) or dorsal skin (B) of mice. mRNA expression of α-Sma, Col1a1 and Col1a2 in the ear was measured 3 or 5 days after final TGF-β administration was evaluated. Gene expression levels (normalized to Gapdh) are expressed as the fold change of vehicle-treated control. Values are mean ± SE (n = 2–5). #p<0.05 compared to PBS-injected normal mice, *p<0.05 compared to PBS-injected normal mice.
Fig 6.
Effects of topical T-3833261 or Halofuginone on TGF-β-induced fibrotic genes expression in mouse ear skin.
TGF-β (500 ng/20 μL) or vehicle was intradermally injected 5 times in the ear of mice. T-3833261, Halofuginone or vehicle was locally administered to the mice 1 h before TGF-β injection. Skin biopsies were taken from each sample treated mice. mRNA expression of α-Sma, Col1a1 and Col1a2 in the ear was measured 78 h after final TGF-β administration was evaluated. Gene expression levels (normalized to Gapdh) are expressed as the fold change of vehicle-treated control. Values are mean ± SE (n = 4–8). #p<0.05 compared to PBS-injected normal mice, *p<0.05 compared to TGF-β-injected mice.
Fig 7.
Effects of proline addition on PRS inhibitors (T-3833261 or Halofuginone)-induced reduction of α-SMA mRNA expression in skin fibroblasts.
Skin fibroblasts were treated with or without T-3833261 or Halofuginone (1–300 nM) and/or proline (0.05, 0.2 and 1 mM) for 24 h. After incubation for 24 h, mRNA levels were measured by quantitative real-time RT-PCR. Gene expression levels (normalized to GAPDH) are expressed as the fold change of vehicle-treated control. Values are means ± SD (n = 3).