TGF-β1 belongs to a large super-family of multifunctional polypeptide factors. The TGF-β family includes three genes, TGFβ1, TGFβ2 and TGFβ3, which are pleiotropic modulators of cell growth and differentiation, embryonic and bone development, extracellular matrix formation, hematopoiesis, immune and inflammatory responses. These genes have high homology with one another. In mammals, the TGFβ super-family includes various TGFβ genes, as well as the embryonic morphogenes, such as the family of the activins, inhibins, “Mullerian Inhibiting Substance”, and bone morphogenic protein (BMP). Roberts and Sporn, The Transforming Growth Factor-βs in Peptide Growth Factors and Their Receptors. I. Handbook of Experimental Pharmacology, vol. 95/I, Springer-Verlag, Berlin, 419–472 (1990). Each member of the TGF-β family exerts a wide range of biological effects on a large variety of cell types, e.g., they regulate cell growth, morphogenesis, differentiation, matrix production and apoptosis. Lagna et al., Nature, 383:832–836 (1996). TGF-β acts as a growth inhibitor for many cell types and appears to play a central role in the regulation of embryonic development, tissue regeneration, immuno-regulation, as well as in fibrosis and carcinogenesis. TGFβ1 inhibits the growth of many cell types, including epithelial cells, but stimulates the proliferation of various types of mesenchymal cells.
In addition, TGFβs induce the synthesis of extracellular matrix (ECM) proteins, modulate the expression of matrix proteinases and proteinase inhibitors and change the expression of integrins. ECM is a dynamic superstructure of self aggregating macromolecules including fibronectin, collagen and proteoglycan. ECM is the chief pathologic feature of fibrotic diseases. ECM disorder has been proposed to play a central role in pathogenesis disorders such as hypertensive vascular disease and diabetic renal disease. Sato et al., Am. J. Hypertens., 8:160–166 (1995); Schulick et al., Proc. Natl. Acad. Sci., 95:6983–6988 (1988). Moreover, TGFβs are expressed in large amounts in many tumors. Derynck, Trends Biochem. Sci., 19:548–553, (1994). This strong occurrence in neoplastic tissues could indicate that TGFβs are strategic growth/morphogenesis factors which influence the malignant properties associated with the various stages of the metastatic cascade. TGFβs inhibit the growth of normal epithelial and relatively differentiated carcinoma cells, whereas undifferentiated tumor cells which lack many epithelial properties are generally resistant to growth inhibition by TGFβs (Hoosein et al., Exp. Cell. Res. 181:442–453(1989); Murthy et al., Int'l J. Cancer, 44:110–115(1989). Furthermore TGFβ1 may potentiate the invasive and metastatic potential of a breast adenoma cell line (Welch et al., Proc. Natl. Acad. Sci., 87:7678–7682(1990), which indicates a role of TGFβ1 in tumor progression. The molecular mechanisms underlying the effect of TGFβs during tumor cell invasion and metastasization do, however, require further explanation.
The cellular effects of TGFβ are exerted by ligand-induced hetero-oligomerization of two distantly related type I and type II serine/threonine kinase receptors, TGFβR-I and TGFβ R-II, respectively. Lin and Lodish, Trends Cell Biol., 11:972–978. (1993); Massague and Weis-Garcia, Cancer Surv., 27:41–64(1996); ten Dijke et al., Curr. Opin. Cell Biol., 8:139–145 (1996). The two receptors, both of which are required for signaling, act in sequence; TGFβR-I is a substrate for the constitutively active TGFβR-II kinase. Wrana et al., Nature, 370:341–347 (1994); Wieser et al., EMBO J., 14:2199–2208 (1995). Upon TGFβ1 binding, the type II receptor phosphorylates threonine residues in the GS domain of ligand occupied type I receptor or activin-like kinase (ALK5), which results in activation of type I receptors. The TGFβ1 type I receptor in turn phosphorylates Smad2 and Smad3 proteins which translocate to the nucleus and mediate intracellular signaling. The inhibition of ALK5 phosphorylation of Smad3 will reduce TGFβ1 induced extracellular matrix production. Krettzchmar et al., Genes Dev., 11: 984–995 (1997); Wu et al., Mol. Cell. Biol., 17:2521–2528 (1997); U.S. Pat. No. 6,465,493.
TGFβ is a powerful and essential immune regulator in the vascular system capable of modulating inflammatory events in both leuko and vascular endothelial cells. Shull et al., Nature, 359:693–699 (1992). It is also involved in the pathogenesis of chronic vascular diseases such as atherosclerosis and hypertension. Grainger & Metcalfe et al., Bio. Rev. Cambridge Phil. Soc., 70:571–596 (1995); Metcalfe et al., J. Human Hypertens., 9:679 (1995).
Genetic studies of TGFβ-like signaling pathways in Drosophila and Caenorhabditis elegans have led to the identification of mothers against dpp (Mad). Sekelsky et al., Genetics, 139:1347–1358 (1995) and sma genes respectively. Savage et al., Proc. Natl. Acad. Sci. USA, 93:790–794, (1996). The products of these related genes perform essential functions downstream of TGFβ-like ligands acting via serine/threonine kinase receptors in these organisms. Wiersdorf et al., Development, 122:2153–2163 (1996); Newfeld et al., Development, 122:2099–2108 (1996); Hoodless et al., Cell, 85:489–500 (1996). Vertebrate homologs of Mad and sma have been termed Smads. Derynck et al., Cell, 87:173 (1996) or MADR genes. Wrana and Attisano, Trends Genet., 12:493–496 (1996). SMAD proteins have been identified as signaling mediators of TGFβ superfamily. Hahn et al., Science, 271:350–353 (1996). Genetic alterations in Smad2 and Smad4/DPC4 have been found in specific tumor subsets, and thus Smads may function as tumor suppressor genes. Hahn et al., Science, 271:350–353 (1996); Riggins et al., Nature Genet., 13:347–349 (1996); Eppert et al., Cell, 86:543–552 (1996). Smad proteins share two regions of high similarity, termed MH1 and MH2 domains, connected with a variable proline-rich sequence. Massague, Cell, 85:947–950 (1996); Derynck and Zhang, Curr. Biol., 6:1226–1229 (1996). The C-terminal part of Smad2, when fused to a heterologous DNA-binding domain, was found to have transcriptional activity. Liu et al., Nature, 381:620–623 (1996); Meersseman et al., Mech. Dev., 61:127–140 (1997). The intact Smad2 protein when fused to a DNA-binding domain, was latent, but transcriptional activity was unmasked after stimulation with ligand. Liu et al., supra.
TGFβ initiates an intracellular signaling pathway leading ultimately to the expression of genes that regulate the cell cycle, control proliferative responses, or relate to extracellular matrix proteins that mediate outside-in cell signaling, cell adhesion, migration and intercellular communication.
There exists a need for effective therapeutic agents for inhibiting TGFβ activity, as well as for inhibiting the phosphorylation of smad2 or smad3 by TGFβ type I or activin-like kinase (ALK5) receptor and for preventing and treating disease states mediated by the TGFβ signaling pathway in mammals. In particular, there continues to be a need for compounds that selectively inhibit TGFβ, especially the ALK5 receptor.