Indole-derived compound SIS3 targets a subset of activated Smad complexes

Smad2 and Smad3 are receptor-regulated Smad proteins that transmit signals from cytokines belonging to the transforming growth factor (TGF)-β family, which are vital for adult tissue homeostasis. The overactivation of such proteins often engenders the development of pathological conditions. Smad3 reportedly mediates TGF-β-induced fibrosis. Although various potential Smad3-specific inhibitors are being developed, their specificity and action mechanisms remain largely unknown. This study aimed to establish a biochemical platform to monitor Smad2- or Smad3-dependent TGF-β signaling using SMAD2, SMAD3 and SMAD2/3 knockout cell lines alongside TGF-β-dependent luciferase reporters and Smad mutant proteins. Using this platform, SIS3, an indole-derived compound widely used as a specific Smad3 inhibitor, was observed to preferentially suppress a subset of activated Smad complexes. However, its inhibition did not favor Smad3 signaling over Smad2 signaling. These findings indicate that SIS3 can be employed as a probe to examine the heterogeneous nature of Smad signaling that induces gene expression. However, its use as a Smad3-specific inhibitor should be avoided.