In vitro activity: TP0427736 is a novel, potent and selective inhibitor of ALK5 kinase with IC50 value of 2.72 nM and appropriate in vitro and in vivo profiles. The inhibitory effect on ALK5 kinase is 300-fold higher than thaton ALK3 (IC50 = 836 nM for ALK3). It also inhibits Smad2/3 phosphorylation in A549 cells induced by TGF-β1 with an IC50 value of 8.68 nM. TP0427736 inhibited ALK5 kinase activity with an IC50 of 2.72nM; this effect was 300-fold higher than the inhibitory effect on ALK3. In cell-based assays, TP0427736 inhibited Smad2/3 phosphorylation in A549 cells and decreased the growth inhibition of human outer root sheath cells. The topical application of TP0427736 significantly decreased Smad2 phosphorylation in mouse skin, and its repeated application suppressed the shortening of average hair follicle length during the transition from the late anagen phase to the catagen phase. TP0427736 may serve as a potential new therapy for AGA (Androgenic alopecia), which occurs as a result of the contraction of the anagen phase because of the action of androgens on hair follicles. TGF-β production from dermal papillae is enhanced by androgens, and growth inhibition of hair-follicle cells is induced by TGF-β, and the hair cycle progresses from the anagen phase to the catagen phase.

Kinase Assay: TP0427736 inhibits Smad2/3 phosphorylation in a concentration-dependent manner, with an IC50 value of 8.68 nM. In a cell-based assay of outer root sheath cells, TP0427736 rescued the inhibited proliferation by TGF-β1 and TGF-β2 in a concentration-dependent manner

Cell Assay: A549 cells are cultured in plates overnight at 37℃ in a 5% CO2, 95% air atmosphere. Cells are pretreated with various concentrations of TP0427736 or DMSO as a control for 2 h, followed by the addition of 1 ng/mL TGF-β1. After 1 h of incubation, the cells are washed with PBS and lysed with RIPA solution, then mixed with biotinylated anti-Smad2/3 antibody. The mixture is transferred to a streptavidin-coated 96 well plate and incubated for 2 h. The mixture is then discarded, and each well is treated with rabbit anti-phosphoserine antibody, followed by Eu-labeled anti-rabbit IgG antibody and DELFIA Enhancement solution. The developed fluorescence is measured using an ARVO multi-label counter. The IC50 values are determined by analyzing the concentration-response curves.