Inhibition of gene markers of fibrosis with a novel inhibitor of transforming growth factor-β type I receptor kinase in puromycin-induced nephritis

SB-525334 ( 6-[ 2- tert- butyl- 5-( 6- methyl- pyridin-2-yl)-1H-imidazol-4- yl]- quinoxaline) has been characterized as a potent and selective inhibitor of the transforming growth factor-beta 1 ( TGF-beta 1) receptor, activin receptor- like kinase ( ALK5). The compound inhibited ALK5 kinase activity with an IC50 of 14.3 nM and was similar to 4- fold less potent as an inhibitor of ALK4 ( IC50 = 58.5 nM). SB-525334 was inactive as an inhibitor of ALK2, ALK3, and ALK6 ( IC50 > 10,000 nM). In cell-based assays, SB-525334 ( 1 mu M) blocked TGF-beta 1-induced phosphorylation and nuclear translocation of Smad2/3 in renal proximal tubule cells and inhibited TGF-beta 1-induced increases in plasminogen activator inhibitor-1 ( PAI-1) and procollagen alpha 1( I) mRNA expression in A498 renal epithelial carcinoma cells. In view of this profile, SB-525334 was used to investigate the role of TGF-beta 1 in the acute puromycin aminonucleoside ( PAN) rat model of renal disease, a model of nephritis-induced renal fibrosis. Orally administered doses of 1, 3, or 10 mg/kg/day SB- 525334 for 11 days produced statistically significant reductions in renal PAI-1 mRNA. Also, the compound produced dose-dependent decreases in renal procollagen alpha 1( I) and procollagen alpha 1( III) mRNA, which reached statistical significance at the 10- mg/ kg/ day dose when compared with vehicle-treated PAN controls. Furthermore, PAN-induced proteinuria was significantly inhibited at the 10- mg/ kg/ day dose level. These results provide further evidence for the involvement of TGF-beta 1 in the profibrotic changes that occur in the PAN model and for the first time, demonstrate the ability of a small molecule inhibitor of ALK5 to block several of the markers that are predictive of fibrosis and renal injury in this model.