Antifibrotic activity of a FIEL1 small molecule inhibitor in vitro. (A) Structural analysis of the FIEL1 HECT domain revealed a major cavity within the C terminus of the HECT domain. (B) Structures of the BC-1480 backbone (4-(2-Oxo-2,3-dihydro-1H-benzoimidazole-5-sulfonylamino)-benzoic acid) and lead compound BC-1485. (C and D) Docking studies of the lead compound, BC-1485, interacting with the FIEL1-HECT domain. (E) FIEL1 protein was HIS-purified from FIEL1 expression in 293T cells using cobalt beads. Beads were then extensively washed before exposure to BC-1480 or BC-1485 at different concentrations (10−4 to 100 µM). Purified PIAS4 protein was then incubated with drug-bound FIEL1 beads overnight. Beads were washed, and proteins were eluted and resolved on SDS-PAGE. The relative amounts of PIAS4 detected in the pull-downs was normalized to loading and quantified (n = 2). (F) In vitro ubiquitination assay. Purified FIEL1, E1, and E2 protein were incubated with purified V5-PIAS4, and the full complement of ubiquitination reaction components with increased concentrations of BC-1485 showed decreased levels of polyubiquitinated PIAS4 (arrows). (bottom) Levels of ubiquitinated PIAS4 as a function of BC-1485 concentration (n = 2). (G) MLE cells were exposed to BC-1480 or BC-1485 at various concentrations for 18 h. Cells were then collected and immunoblotted (n = 3). (H) PIAS4 protein half-life determination after BC-1480 or BC-1485 treatment at 5 µM for 18 h. (I) PIAS4 and FIEL1 mRNA analysis after BC-1485 treatment for 18 h. Data represent mean values ± SEM (n = 3 independent experiments; NS, not significant compared with 0 µM condition, Student’s t test).
