SB-431542 Is a Potent and Specific Inhibitor of Transforming Growth Factor- Superfamily Type I Activin Receptor-Like Kinase (ALK) Receptors ALK4, ALK5, and ALK7

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. TGFsuperfamily 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 TGFtype 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 TGFsignaling 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. The TGFsuperfamily is a large family of growth and differentiation factors that regulate a wide variety of cellular processes in many different cell types and biological contexts. Different family members regulate cell proliferation (both positively and negatively), migration, extracellular matrix elaboration, adhesion, survival and differentiation, in both developing embryos and adult organisms, ranging from worms to humans (Whitman, 1998; Massagué and Chen, 2000; Massagué et al., 2000). Aberrant signaling by TGF, the prototype of the family, has been implicated in a number of human diseases, including cancer, hereditary hemorrhagic telangiectasia, atherosclerosis, and fibrotic disease of the kidney, liver, and lung (Blobe et al., 2000). In addition, low levels of TGFsignaling have been implicated in compromised wound healing, and inappropriately high levels of TGFsignaling are associated with excessive scarring (Roberts and Sporn, 1993). The mechanism of signaling by TGFfamily members is now understood in some detail. The ligands bring together a type II receptor with a type I receptor, both serine/threonine kinases. The type II receptor phosphorylates and activates the type I receptor in the complex. To date, there are five mammalian type II receptors: T R-II, ActR-II, ActR-IIB, This work was funded by Imperial Cancer Research Fund (now Cancer Research UK after the merger of Imperial Cancer Research Fund with the Cancer Research Campaign), GlaxoSmithKline Pharmaceuticals, and a Medical Research Council training fellowship (to F.J.N.). G.J.I. and F.J.N. contributed equally to this work. ABBREVIATIONS: TGF, transforming growth factor ; BMP, bone morphogenetic protein; AMH, anti-Müllerian hormone; ALK, activin receptor-like kinase; SB-431542, 4-(5-benzo[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide; DE, distal element; OP, operator; SRF, serum response factor; FCS, fetal calf serum; BSA, bovine serum albumin; EGF, epidermal growth factor; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulfoxide; GRB2, growth-factor receptor-bound protein 2; ATF, activating transcription factor; SRE, serum response element; -Gal, -galactosidase; GS, glycineand serine-rich. 0026-895X/02/6201-65–74$7.00 MOLECULAR PHARMACOLOGY Vol. 62, No. 1 Copyright © 2002 The American Society for Pharmacology and Experimental Therapeutics 1516/989400 Mol Pharmacol 62:65–74, 2002 Printed in U.S.A. 65 at A PE T Jornals on Jauary 7, 2018 m oharm .aspeurnals.org D ow nladed from BMPR-II, and AMHR-II and seven type I receptors (ALKs 1–7; Piek et al., 1999). In most cell types, TGFsignals through the combination of T R-II and ALK5 (Piek et al., 1999); in endothelial cells, however, ALK1 acts as a TGFtype I receptor (Oh et al., 2000). Activin and related ligands signal via combinations of ActR-II or ActR-IIB and ALK4, and BMPs signal through combinations of ALK2, ALK3, and ALK6 with ActR-II, ActR-IIB, or BMPR-II (Piek et al., 1999). AMH signals through a complex of AMHR-II with ALK6 (Gouedard et al., 2000), and nodal has been shown recently to signal through a complex of ActR-IIB and ALK7 (Reissmann et al., 2001). The signals are transduced to the nucleus primarily through activation of complexes of Smads. Upon activation, the type I receptors phosphorylate members of the receptorregulated subfamily of Smads at two serines in an SSXS motif at their extreme C termini. This activates them and enables them to form complexes with a common mediator Smad, Smad4 (Piek et al., 1999). Smads 1, 5, and 8 are substrates for ALKs 1, 2, 3, and 6, whereas Smads 2 and 3 are substrates for ALKs 4, 5, and 7 (Piek et al., 1999; Jornvall et al., 2001). The activated Smad complexes accumulate in the nucleus, where they are directly involved in the transcription of target genes, usually in association with other specific DNA-binding transcription factors (Massagué and Wotton, 2000). In addition, TGFsuperfamily members can also induce the activation of all three known MAP kinase pathways, although the mechanism underlying this remains unclear (Massagué and Chen, 2000). Small molecule inhibitors have been invaluable in other systems for dissecting the mechanisms of signal transduction pathways and understanding the role of individual signaling pathways in different biological processes. In addition, they have the potential to be useful for therapeutic applications (Blake et al., 2000 and references therein). Compounds that specifically inhibit receptor kinases for TGFsuperfamily members would be enormously beneficial for furthering our understanding of the mechanism of signaling and determining which biological processes require these signaling pathways. Compounds that selectively inhibit the receptors for TGF, in particular, have the potential to be developed for therapeutic applications in the treatment of fibrosis, latestage carcinogenesis, atherosclerosis, and excessive scarring (i.e., diseases in which the activity of the TGFsignaling pathway has been implicated). A potent inhibitor of ALK5 (SB-431542) has recently been developed that acts as a competitive ATP binding site kinase inhibitor and has been shown to inhibit the in vitro phosphorylation of immobilized Smad3 with an IC50 of 94 nM (compound 14; Callahan et al., 2002). We have now investigated the efficiency of SB-431542 as an ALK5 inhibitor and rigorously tested its specificity. We demonstrate that, of the ALKs, it inhibits the activity of ALK5 and also ALK4 and ALK7, which are very similar to ALK5 in their kinase domains. It does not significantly inhibit any of the other ALKs, which have more divergent kinase domains. Consistent with this, SB-431542 inhibits TGFand activin-induced phosphorylation of Smad2, which is mediated by ALK5 and ALK4, respectively, but not BMPinduced phosphorylation of Smad1, which is mediated by ALKs 2, 3, and 6. To demonstrate the specificity of SB-431542 for ALKs 4, 5, and 7, we have tested its effect on several other signaling pathways whose activities depend on the concerted activation of multiple kinases. SB-431542 had no effect on any of these signaling pathways, demonstrating that it is highly selective for these ALKs. Materials and Methods Plasmids. The following plasmids have been described previously: constitutively active human ALK1, ALK3, ALK4, ALK5, ALK6, and rat ALK7 in mammalian expression vectors (Nakao et al., 1997; Macias-Silva et al., 1998; Pierreux et al., 2000; Jornvall et al., 2001), wild-type human ALK4, ALK5, and ALK7 in mammalian expression vectors (ten Dijke et al., 1994; Jornvall et al., 2001), EF-Flag Mixer and EF-Flag XSmad2 (Germain et al., 2000), EFLacZ (Bardwell and Treisman, 1994), DE-driven luciferase reporter plasmid (Pierreux et al., 2000), Lex-OP-luciferase (Gineitis and Treisman, 2001), mammalian expression plasmids encoding NLex.ElkC (Marais et al., 1993) and NLex.JunN (Price et al., 1996), EF-MEKK1 and EF-RasV12 (Price et al., 1995), and mammalian expression plasmid encoding Flag-tagged constitutively active MKK3 (Raingeaud et al., 1996). The CAGA12-luciferase reporter gene consists of 12 tandem copies of the “CAGA” Smad binding element (Dennler et al., 1998) upstream of the adenovirus major late promoter driving luciferase gene expression. 3D.A-luciferase was constructed by moving the three SRF binding sites and Xenopus laevis minimal -actin promoter from 3D.A-Fos (Mohun et al., 1987) into pGL3 (Promega, Madison, WI). EF-Flag XSmad1 was constructed by subcloning X. laevis Smad1 into EF-Flag (Germain et al., 2000). Constitutively active mouse ALK2 was constructed by subcloning the ALK2 coding sequence containing the Q207D mutation (Armes and Smith, 1997) into an EF expression vector. Cell Culture, Transfections, Inductions, and Inhibitors. HaCaT, NIH 3T3, C2C12, and T47D cells were all maintained in DMEM containing 10% FCS. NIH 3T3 cells were transfected using LipofectAMINE (Invitrogen, Carlsbad, CA). Recombinant human TGF1 (PeproTech Inc., Rocky Hill, NJ) was dissolved in 4 mM HCl/1 mg/ml BSA at a concentration of 1 g/ml and was used at a final concentration of 2 ng/ml. Activin was dissolved in 1 mg/ml BSA in phosphate-buffered saline and used at a concentration of 10 to 20 ng/ml. BMP4 (R & D Systems, Minneapolis, MN) was dissolved in 1 mg/ml BSA in phosphate-buffered saline and used at 20 ng/ml. EGF (R & D Systems) was dissolved in 10 mM acetic acid/0.1% BSA and used at 30 ng/ml. Osmotic shock was performed by incubating cells in 0.7 M NaCl in DMEM for 20 min. Solid anhydrous SB-431542 was dissolved at a concentration of 10 mM in DMSO. Further dilutions of SB-431542 in DMSO were made so that in all cases, SB-431542 was added to cells from a 1000 stock. U0126 (Promega) was dissolved in DMSO and used at a concentration of 25 M. Kinase Assays, Whole-Cell Extracts, Western Blotting, and Transcriptional Assays. Kinase assays were performed as described previously (Laping et al., 2002). For the Western blots shown in Fig. 4, extracts were made using 20 mM HEPES, pH 7.5, 10% glycerol, 400 mM KCl, 2 mM EDTA, 1% Triton, 1 mM dithiothreitol, 25 mM NaF, 25 mM sodium-glycerophosphate, 1 mM Na3VO4, and protease inhibitors. For all other Western blots, extracts were made using the whole-cell extraction buffer described previously (Khwaja et al., 1998). Western blotting was performed using standard techniques. The following antibodies were used: monoclonal antibody against Smad2 (which also recognizes Smad3; Transduction Laboratories, Lexington, KY); monoclonal antibody against Smad1 [A4 (Santa Cruz Biotechnology, Santa Cruz, CA), Fig. 1, or MADR1 (Upstate, Inc., Lake Placid, NY), Fig. 4]; polyclonal antibodies against phosphorylated Smad1 and Smad2 (a kind gift from Peter ten Dijke) (Faure et al., 2000); monoclonal antibody against GRB2 (Transduction Laboratories); polyclonal antibodies against pan ERK, pan p38, phosphorylated JNK, phosphorylated p38, and phosphorylated ATF2, and a monoclonal antibody against phosphorylated 66 Inman et al. at A PE T Jornals on Jauary 7, 2018 m oharm .aspeurnals.org D ow nladed from ERK1 and ERK2 (New England Biolabs UK, Hitchin, UK). Transcriptional assays for luciferase reporter genes were performed as described previously (Pierreux et al., 2000).