Small molecule inhibitors have proven extremely useful for investigating signal transduction pathways and have the potential for development into therapeutics for inhibiting signal transduction pathways whose activities contribute to human diseases. Transforming growth factor ␤ (TGF-␤) is a member of a large family of pleiotropic cytokines that are involved in many biological processes, including growth control, differentiation, migration, cell survival, adhesion, and specification of developmental fate, in both normal and diseased states. TGF-␤ superfamily members signal through a receptor complex comprising a type II and type I receptor, both serine/threonine kinases. Here, we characterize a small molecule inhibitor (SB-431542) that was identified as an inhibitor of activin receptor-like kinase (ALK)5 (the TGF-␤ type I receptor). We demonstrate that it inhibits ALK5 and also the activin type I receptor ALK4 and the nodal type I receptor ALK7, which are very highly related to ALK5 in their kinase domains. It has no effect on the other, more divergent ALK family members that recognize bone morphogenetic proteins (BMPs). Consistent with this, we demonstrate that SB-431542 is a selective inhibitor of endogenous activin and TGF-␤ signaling but has no effect on BMP signaling. To demonstrate the specificity of SB-431542, we tested its effect on several other signal transduction pathways whose activities depend on the concerted activation of multiple kinases. SB-431542 has no effect on components of the ERK, JNK, or p38 MAP kinase pathways or on components of the signaling pathways activated in response to serum.

Transforming growth factor ␤1 (TGF-␤1) is a potent fibrotic factor responsible for the synthesis of extracellular matrix. TGF-␤1 acts through the TGF-␤ type I and type II receptors to activate intracellular mediators, such as Smad proteins, the p38 mitogen-activated protein kinase (MAPK), and the extracellular signal-regulated kinase pathway. We expressed the kinase domain of the TGF-␤ type I receptor [activin receptor-like kinase (ALK)5] and the substrate, Smad3, and determined that SB-431542 is a selective inhibitor of Smad3 phosphorylation with an IC 50 of 94 nM. It inhibited TGF-␤1-induced nuclear Smad3 localization. The p38 mitogen-activated protein kinase inhibitors SB-203580 and SB-202190 also inhibit phosphorylation of Smad3 by ALK5 with IC 50 values of 6 and 3 M, respectively. This suggests that these p38 MAPK inhibitors must be used at concentrations of less than 10 M to selectively address p38 MAPK mechanisms. However, the p38 MAPK inhibitor SB-242235 did not inhibit ALK5. To evaluate the relative contribution of Smad signaling and p38 MAPK signaling in TGF-␤1-induced matrix production, the effect of SB-431542 was compared with that of SB-242235 in renal epithelial carcinoma A498 cells. All compounds inhibited TGF-␤1-induced fibronectin (FN) mRNA, indicating that FN synthesis is mediated in part via the p38 MAPK pathway. In contrast, SB-431542, but not the selective p38 MAPK inhibitor SB-242235, inhibited TGF-␤1-induced collagen I␣1 (col I␣1). These data indicate that some matrix markers that are stimulated by TGF-␤1 are mediated via the p38 MAPK pathway (i.e., FN), whereas others seem to be activated via ALK5 signaling independent of the p38 MAPK pathway (i.e., col I␣1).