Synthesis and biological evaluation of novel 2-pyridinyl-[1,2,3]triazoles as inhibitors of transforming growth factor beta 1 type 1 receptor

A series of 2-pyridinyl-[1,2,3]triazoles have been synthesized and evaluated for their ALK5 inhibitory activity in the luciferase reporter assays. Compound 8d showed significant ALK5 inhibition (SBE-luciferase activity, 25%; p3TP-luciferase activity, 17%) at a concentration of 5 microM that is comparable to that of SB-431542 (SBE-luciferase activity, 21%; p3TP-luciferase activity, 12%), but weak p38 alpha MAP kinase inhibition (13%) at a concentration of 10 microM that is much lower than that of SB-431542 (54%).     

Activation and roles of ALK4/ALK7-mediated maternal TGFbeta signals in zebrafish embryo

Activin, Nodal, and Vg1, members of the transforming growth factor beta (TGFbeta) superfamily, transduce signal through type I receptors ALK4 or ALK7 and play important roles in mesoderm induction and patterning during vertebrate embryogenesis. However, the timing and magnitude of the ALK4/ALK7-mediated maternal TGFbeta signals are not clear. SB-431542 is identified as an inhibitor of the ALK4/ALK5/ALK7-mediated TGFbeta signals and its specificity in vertebrate embryos has not been reported. We demonstrate that SB-431542 is able to specifically and reproducibly block the Smad2/3-mediated TGFbeta signals in zebrafish embryo. Embryos exposed to SB-431542 exhibit various defects phenocopying Nodal-deficient mutants. SB-431542 treatments starting at different cell cycles before the midblastula transition lead to different degrees of developmental defects in mesoderm induction and patterning, suggesting that maternal TGFbeta signals are activated right after fertilization and required for mesoderm formation and patterning.     

ALK5 inhibition blocks TGFβ-induced CCN1 expression in human foreskin fibroblasts

The potent profibrotic cytokine TGFβ induces connective tissue growth factor (CCN2/CTGF) is induced in fibroblasts in a fashion sensitive to SB-431542, a specific pharmacological inhibitor of TGFβ type I receptor (ALK5). In several cell types, TGFβ induces CCN1 but suppresses CCN3, which opposes CCN1/CCN2 activities. However, whether SB-431542 alters TGFβ-induced CCN1 or CCN3 in human foreskin fibroblasts in unclear. Here we show that TGFβ induces CCN1 but suppresses CCN3 expression in human foreskin fibroblasts in a SB-431542-sensitive fashion. These results emphasize that CCN1/CCN2 and CCN3 are reciprocally regulated and support the notion that blocking ALK5 or addition of CCN3 may be useful anti-fibrotic approaches.     

Oocyte-secreted factor activation of SMAD 2/3 signaling enables initiation of mouse cumulus cell expansion

Expansion of the mouse cumulus-oocyte complex (COC) is dependent on oocyte-secreted paracrine factors. Transforming growth factor beta (TGFB) superfamily molecules are prime candidates for the cumulus expansion-enabling factors (CEEFs), and we have recently determined that growth differentiation factor 9 (GDF9) alone is not the CEEF. The aim of this study was to examine oocyte paracrine factors and their signaling pathways that regulate mouse cumulus expansion. Using RT-PCR, oocytes were found to express the two activin subunits, Inhba and Inhbb, and activin A and activin B both enabled FSH-induced cumulus expansion of oocytectomized (OOX) complexes. Follistatin, an activin-binding protein, neutralized activin-induced expansion but had no effect on oocyte-induced expansion. The type I receptors for GDF9 and activin are activin receptor-like kinase 5 (ALK5) and ALK4, respectively, both of which activate the same SMAD 2/3 signaling pathway. We examined the requirement for this signaling system using an ALK 4/5/7 inhibitor, SB-431542. SB-431542 completely ablated FSH-stimulated GDF9-, activin A-, activin B-, and oocyte-induced cumulus expansion. Moreover, SB-431542 also antagonized epidermal growth factor-stimulated, oocyte-induced cumulus expansion. Using real-time RT-PCR, SB-431542 also attenuated GDF9-, activin A-, and oocyte-induced OOX expression of hyaluronan synthase 2, tumor necrosis factor alpha-induced protein 6, prostaglandin synthase 2, and pentraxin 3. This study provides evidence that the CEEF is composed of TGFB superfamily molecules that signal through SMAD 2/3 to enable the initiation of mouse cumulus expansion.     

Smad and p38 MAP kinase-mediated signaling of proteoglycan synthesis in vascular smooth muscle

Atherosclerosis is the underlying pathological process of most cardiovascular disease. A critical component of the "response to retention" hypothesis of atherogenesis is proteoglycan/low density lipoprotein (LDL) binding. Transforming growth factor beta (TGF-beta) is present in atherosclerotic lesions, regulates vascular smooth muscle cell (VSMC) proteoglycan synthesis via an unknown signaling pathway, and increases proteoglycan/LDL binding. This pathway was investigated using the activin receptor-like kinase 5 (ALK5) inhibitor SB431542 and inhibitors of p38 MAP kinase as a possible downstream or alternative mediator. TGF-beta stimulated and SB431542 inhibited the phosphorylation of Smad2/3. In human VSMC, TGF-beta increased [(35)S]sulfate incorporation into proteoglycans associated with a 19% increase in glycosaminoglycan (GAG) chain size by size exclusion chromatography. SB431542 caused a concentration-dependent decrease in TGF-beta-mediated [(35)S]sulfate incorporation with 92% inhibition at 3 mum. Two different p38 MAP kinase inhibitors, SB203580 and SB202190, but not the inactive analogue SB202474, concentration-dependently blocked TGF-beta-mediated [(35)S]sulfate incorporation. TGF-beta increased [(3)H]glucosamine incorporation into glycosaminoglycans by 180% and [(35)S]Met/Cys incorporation into proteoglycan core proteins by 35% with both effects completely inhibited by SB431542. Blocking both Smad2/3 and p38 MAP kinase pathways prevented the effect of TGF-beta to increase proteoglycan to LDL binding. TGF-beta mediates its effects on proteoglycan synthesis in VSMCs via the ALK5/Smad2/3 phosphorylation pathway as well as via the p38 MAP kinase signaling cascade. Further studies of downstream pathways controlling proteoglycan synthesis may identify potential therapeutic targets for the prevention of atherosclerosis and cardiovascular disease.     

Inhibitor-resistant type I receptors reveal specific requirements for TGF-beta signaling in vivo

Activin/nodal-like TGF-beta superfamily ligands signal through the type I receptors Alk4, Alk5, and Alk7, and are responsible for mediating a number of essential processes in development. SB-431542, a chemical inhibitor of activin/nodal signaling, acts by specifically interfering with type I receptors. Here, we use inhibitor-resistant mutant receptors to examine the efficacy and specificity of SB-431542 in Xenopus and zebrafish embryos. Treatment with SB-431542 eliminates Smad2 phosphorylation in vivo and generates a phenotype very similar to those observed in genetic mutants in the nodal signaling pathway. Inhibitor-resistant Alk4 efficiently rescues Smad2 signaling, developmental phenotype, and marker gene expression after inhibitor treatment. This system was used to examine type I receptor specificity for several activin/nodal ligands. We find that Alk4 can efficiently rescue signaling by a wide range of ligands, while Alk7 can only weakly rescue signaling by the same ligands. In whole embryos, nodal signaling during gastrulation can be rescued with Alk4, but not Alk7, while Alk5 can only mediate signaling by ligands expressed later in development. The combination of the ALK inhibitor SB-431542 with inhibitor-resistant ALKs provides a powerful set of tools for examining nodal/activin signaling during embryogenesis.     

Synthesis and biological evaluation of 1-(6-methylpyridin-2-yl)-5-(quinoxalin-6-yl)-1,2,3-triazoles as transforming growth factor-β type 1 receptor kinase inhibitors

A series of 1-(6-methylpyridin-2-yl)-5-(quinoxalin-6-yl)-1,2,3-triazoles has been synthesized and evaluated for their ALK5 inhibitory activity. The 1-(6-methylpyridin-2-yl)-1,2,3-triazoles were assembled by Cu(I)-catalyzed azide–alkyne 1,3-dipolar cycloaddition. Following this, quinoxaline was introduced through Pd-catalyzed direct arylation. The synthesized 1-(6-methylpyridin-2-yl)-5-(quinoxalin-6-yl)-1,2,3-triazoles revealed significant selectivity differences with respect to p38α MAP kinase. In particular, 12k showed 80.8% ALK5 inhibitory activity at a concentration of 10 μM and IC₅₀ value of 4.69 μM, but did not show p38α MAP kinase inhibitory activity (?1.94% inhibition at a concentration of 10 μM).     

Synthesis and biological evaluation of 1,2,4-trisubstituted imidazoles and 1,3,5-trisubstituted pyrazoles as inhibitors of transforming growth factor β type 1 receptor (ALK5)

Two series of nitrogenous heterocycle compounds—1,2,4-trisubstituted imidazoles and 1,3,5-trisubstituted pyrazoles have been synthesized and evaluated for their ALK5 inhibitory activity and cytotoxicity in TGFβ-Smad2 assay and MTT assay, respectively. The ALK4/5/7 inhibitory activity of some compound was also evaluated in ALK4/5/7 autophosphorylation assays. Compounds 6c and 14c showed relatively potent ALK5 inhibition while weak cytotoxicity. At the same time, compounds 6c and 14c display relatively better ALK5 selectivity versus ALK4/ALK7 (nearly 10-fold) compared with SB431542, a well known ALK5 inhibitor. Compound 6g2 proved to be a moderately selective ALK4 inhibitor versus ALK5 and ALK7 (>10-fold). The binding mode of 14c generated by flexible docking study shows that 14c fits well into the site cavity of ALK5 by forming several tight interactions.     

Pluripotency Gene Expression and Growth Control in Cultures of Peripheral Blood Monocytes during Their Conversion into Programmable Cells of Monocytic Origin (PCMO): Evidence for a Regulatory Role of Autocrine Activin and TGF-β

Previous studies have shown that peripheral blood monocytes can be converted in vitro to a stem cell-like cell termed PCMO as evidenced by the re-expression of pluripotency-associated genes, transient proliferation, and the ability to adopt the phenotype of hepatocytes and insulin-producing cells upon tissue-specific differentiation. However, the regulatory interactions between cultured cells governing pluripotency and mitotic activity have remained elusive. Here we asked whether activin(s) and TGF-β(s), are involved in PCMO generation. De novo proliferation of PCMO was higher under adherent vs. suspended culture conditions as revealed by the appearance of a subset of Ki67-positive monocytes and correlated with down-regulation of p21^WAF1 beyond day 2 of culture. Realtime-PCR analysis showed that PCMO express ActRIIA, ALK4, TβRII, ALK5 as well as TGF-β1 and the β_A subunit of activin. Interestingly, expression of ActRIIA and ALK4, and activin A levels in the culture supernatants increased until day 4 of culture, while levels of total and active TGF-β1 strongly declined. PCMO responded to both growth factors in an autocrine fashion with intracellular signaling as evidenced by a rise in the levels of phospho-Smad2 and a drop in those of phospho-Smad3. Stimulation of PCMO with recombinant activins (A, B, AB) and TGF-β1 induced phosphorylation of Smad2 but not Smad3. Inhibition of autocrine activin signaling by either SB431542 or follistatin reduced both Smad2 activation and Oct4A/Nanog upregulation. Inhibition of autocrine TGF-β signaling by either SB431542 or anti-TGF-β antibody reduced Smad3 activation and strongly increased the number of Ki67-positive cells. Furthermore, anti-TGF-β antibody moderately enhanced Oct4A/Nanog expression. Our data show that during PCMO generation pluripotency marker expression is controlled positively by activin/Smad2 and negatively by TGF-β/Smad3 signaling, while relief from growth inhibition is primarily the result of reduced TGF-β/Smad3, and to a lesser extent, activin/Smad2 signaling.     

The specificities of small molecule inhibitors of the TGFß and BMP pathways

Small molecule inhibitors of type 1 receptor serine threonine kinases (ALKs1-7), the mediators of TGFß and BMP signals, have been employed extensively to assess their physiological roles in cells and organisms. While all of these inhibitors have been reported as “selective” inhibitors of specific ALKs, extensive specificity tests against a wide array of protein kinases have not been performed. In this study, we examine the specificities and potencies of the most frequently used small molecule inhibitors of the TGFß pathway (SB-431542, SB-505124, LY-364947 and A-83-01) and the BMP pathway (Dorsomorphin and LDN-193189) against a panel of up to 123 protein kinases covering a broad spectrum of the human kinome. We demonstrate that the inhibitors of the TGFß pathway are relatively more selective than the inhibitors of the BMP pathway. Based on our specificity and potency profile and published data, we recommend SB-505124 as the most suitable molecule for use as an inhibitor of ALKs 4, 5 and 7 and the TGFß pathway. We do not recommend Dorsomorphin, also called Compound C, for use as an inhibitor of the BMP pathway. Although LDN-193189, a Dorsomorphin derivative, is a very potent inhibitor of ALK2/3 and the BMP-pathway, we found that it potently inhibited a number of other protein kinases at concentrations sufficient to inhibit ALK2/3 and its use as a selective BMP-pathway inhibitor has to be considered cautiously. Our observations have highlighted the need for caution when using these small molecule inhibitors to assess the physiological roles of BMP and TGFß pathways.     

A 5-HT7 Heteroreceptor-Mediated Inhibition of [3H]Serotonin Release in Raphe Nuclei Slices of the Rat: Evidence for a Serotonergic–Glutamatergic Interaction

Midbrain slices containing the dorsal and medial raphe nuclei were prepared from rat brain, loaded with [3H]serotonin ([3H]5-HT), superfused, and the electrically induced efflux of radioactivity was determined. The nonselective 5-HT receptor agonist 5-carboxamido-tryptamine (5-CT; 0.001 to 1 microM) inhibited the electrically stimulated [3H]5-HT overflow from raphe nuclei slices (IC50 of 3.34 +/- 0.37 nM). This effect of 5-CT on [3H]5-HT overflow was antagonized by the 5-HT7 receptor antagonist SB-258719 (10 microM) and the 5-HT(1B/1D) antagonist SB-216641 (1 microM), the IC50 values for 5-CT in the presence of SB-258719 and SB-216641 were 94.23 +/- 4.84 and 47.81 +/- 4.66 nM. The apparent pA2 values for SB-258719 and SB-216641 against 5-CT were 6.43 and 7.12, respectively. The inhibitory effect of 5-CT on [3H]5-HT overflow was weakly antagonized by 10 microM of WAY-100635, a 5-HT1A receptor antagonist (IC50 6.65 +/- 0.56 nM, apparent pA2 4.99). The antagonist effect of SB-258719 (10 microM) on 5-CT-evoked [3H]5-HT overflow inhibition was also determined in the presence of 1 microM SB-216641 or 1 microM SB-216641 and 10 microM WAY-100635, and additive interactions were found between the antagonists of 5-HT7 and 5-HT1 receptor subtypes. Addition of the Na+ channel blocker tetrodotoxin (1 microM) in the presence of SB-216641 (1 microM) and WAY-100635 (10 microM) attenuated the inhibitory effect of 5-CT on KCl-induced [3H]5-HT overflow. These findings indicate that 5-CT inhibits [3H]5-HT overflow from raphe nuclei slices of the rat by stimulation of 5-HT7 and 5-HT(1B/1D receptors, whereas the role of 5-HT1A receptors in this inhibition is less pronounced. They also suggest that 5-HT7 receptors are probably not located on serotonergic neurons and thus may serve as heteroreceptors in regulation of 5-HT release in the raphe nuclei. 5-CT (0.1 microM) also inhibited [3H]glutamate release, and SB-258719 (10 microLM) suspended this effect. We therefore speculated that the axon terminals of the glutamatergic cortico-raphe neurons may possess 5-HT7 receptors that inhibit glutamate release, which consequently leads to decreased activity of serotonergic neurons. The postulated glutamatergic-serotonergic interaction in the raphe nuclei was further evidenced by the finding that N-methyl-D-aspartate and AMPA enhanced [3H]5-HT release.     

BMP-7/TGF-β1 signalling in myoblasts: components involved in signalling and BMP-7-dependent blockage of TGF-β-mediated CTGF expression

We and others have recently described the antagonistic role of Bone morphogenetic protein-7 (BMP-7) in TGF-β signalling and myogenic differentiation. To specify the underlying mechanism(s), we here analysed the expression and function of the individual components mediating TGF-β1 and BMP-7 responses. We found that BMP-7 at a concentration of 25 ng/ml induces signalling exclusively via ALK2 and ALK3 leading to the activation of Smad1 and Smad5 and subsequent expression of Id proteins. In contrast, low doses of TGF-β1 (0.1 ng/ml) lead to an exclusive activation of ALK5 and phosphorylation of Smad2 and Smad3 that regulate specific target genes including connective tissue growth factor (CTGF). CTGF is rapidly induced by TGF-β1 already 1h after stimulation and reduced by BMP-7 application. Smad1/Smad5 or Id1/2 overexpression reduced the TGF-β1-mediated expression of CTGF. However, although siRNA-mediated knock down of Alk2/3 or Smad1/5 counteracts the BMP-7 effect on basal CTGF expression there was no consistent reversion of the observed BMP-7 effect on TGF-β1-mediated CTGF expression. Moreover, ALK5 inhibition using the SB431542 inhibitor significantly affected CTGF expression only at later time points whereas ERK1/2 inhibition completely abrogated CTGF expression. These findings point towards a regulatory role of BMP-7 that relies on modulation of Mitogen-activated protein kinases rather than mechanisms that are exclusively driven by differential Smad activation.     

TGF-ß induces Lysyl hydroxylase 2b in human synovial osteoarthritic fibroblasts through ALK5 signaling

Lysyl hydroxylase 2b (LH2b) is known to increase pyridinoline cross-links, making collagen less susceptible to enzymatic degradation. Previously, we observed a relationship between LH2b and osteoarthritis-related fibrosis in murine knee joint. For this study, we investigate if transforming growth factor-beta (TGF-ß) and connective tissue growth factor (CTGF) regulate procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) (gene encoding LH2b) and LH2b expression differently in osteoarthritic human synovial fibroblasts (hSF). Furthermore, we investigate via which TGF-ß route (Smad2/3P or Smad1/5/8P) LH2b is regulated, to explore options to inhibit LH2b during fibrosis. To answer these questions, fibroblasts were isolated from knee joints of osteoarthritis patients. The hSF were stimulated with TGF-ß with or without a kinase inhibitor of ALK4/5/7 (SB-505124) or ALK1/2/3/6 (dorsomorphin). TGF-ß, CTGF, constitutively active (ca)ALK1 and caALK5 were adenovirally overexpressed in hSF. The gene expression levels of PLOD1/2/3, CTGF and COL1A1 were analyzed with Q-PCR. LH2 protein levels were determined with western blot. As expected, TGF-ß induced PLOD2/LH2 expression in hSF, whereas CTGF did not. PLOD1 and PLOD3 were not affected by either TGF-ß or CTGF. SB-505124 prevented the induction of TGF-ß-induced PLOD2, CTGF and COL1A1. Surprisingly, dorsomorphin completely blocked the induction of CTGF and COL1A1, whereas TGF-ß-induced PLOD2 was only slightly reduced. Overexpression of caALK5 in osteoarthritic hSF significantly induced PLOD2/LH2 expression, whereas caALK1 had no effect. We showed, in osteoarthritic hSF, that TGF-ß induced PLOD2/LH2 via ALK5 Smad2/3P. This elevation of LH2b in osteoarthritic hSF makes LH2b an interesting target to interfere with osteoarthritis-related persistent fibrosis.     

Synthesis and biological evaluation of 5-(pyridin-2-yl)thiazoles as transforming growth factor-beta type1 receptor kinase inhibitors

A series of 5-(pyridin-2-yl)thiazoles (14a-l and 15a-l) has been synthesized and evaluated for their ALK5 inhibitory activity in cell-based luciferase reporter assays. Among them, 3-[[5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)thiazol-2-ylamino]methyl]benzamide (15i) and 3-[[5-(6-ethylpyridin-2-yl)-4-(quinoxalin-6-yl)thiazol-2-ylamino]methyl]benzamide (15k) showed more than 95% inhibition at 0.1 microM in luciferase reporter assays using HaCaT cells transiently transfected with p3TP-luc reporter construct and ARE-luciferase reporter construct.     

Structural basis for specificity of TGFβ family receptor small molecule inhibitors

Transforming growth factor-β (TGFβ) receptor kinase inhibitors have a great therapeutic potential. SB431542 is one of the mainly used kinase inhibitors of the TGFβ/Activin pathway receptors, but needs improvement of its EC₅₀ (EC₅₀ = 1 μM) to be translated to clinical use. A key feature of SB431542 is that it specifically targets receptors from the TGFβ/Activin pathway but not the closely related receptors from the bone morphogenic proteins (BMP) pathway. To understand the mechanisms of this selectivity, we solved the crystal structure of the TGFβ type I receptor (TβRI) kinase domain in complex with SB431542. We mutated TβRI residues coordinating SB431542 to their counterparts in activin-receptor like kinase 2 (ALK2), a BMP receptor kinase, and tested the kinase activity of mutated TβRI. We discovered that a Ser280Thr mutation yielded a TβRI variant that was resistant to SB431542 inhibition. Furthermore, the corresponding Thr283Ser mutation in ALK2 yielded a BMP receptor sensitive to SB431542. This demonstrated that Ser280 is the key determinant of selectivity for SB431542. This work provides a framework for optimising the SB431542 scaffold to more potent and selective inhibitors of the TGFβ/Activin pathway.     

Transforming growth factor-beta (TGF-beta) type I receptor/ALK5-dependent activation of the GADD45beta gene mediates the induction of biglycan expression by TGF-beta

We have recently shown that induction of biglycan (BGN) expression by transforming growth factor-beta1 (TGF-beta1) required sequential activation of both Smad and p38 mitogen-activated protein kinase signaling (Ungefroren, H., Lenschow, W., Chen, W.-B., and Kalthoff, H. (2003) J. Biol. Chem. 278, 11041-11049). Here, we have analyzed the receptors through which TGF-beta1 controls expression of BGN and GADD45beta, the latter of which is postulated to link early Smad signaling to delayed activation of p38. Ectopic expression of a dominant-negative mutant of the TGF-beta type II receptor in PANC-1 cells abrogated TGF-beta-induced BGN up-regulation. Similarly, inhibition of the TGF-beta type I receptor/ALK5 with either SB431542 or by enforced stable expression of a kinase-dead mutant greatly attenuated the TGF-beta effect on both BGN and GADD45beta expression in PANC-1 and MG-63 cells. The enhancing effect of ALK5 on TGF-beta-mediated GADD45beta and BGN expression and on GADD45beta promoter activity was also dependent on its ability to activate Smad signaling, because an ALK5 mutant defective in Smad activation (TbetaRImL45) but with an otherwise functional kinase domain failed to mediate these responses. The TGF-beta/ALK5 effect on p38 activation and BGN expression was mimicked by overexpression of GADD45beta alone (in the absence of TGF-beta stimulation) and suppressed upon antisense inhibition of GADD45beta expression. These results show that TGF-beta induces BGN expression through (the Smad-activating function of) ALK5 and GADD45beta and suggest that the sensitivity of MyD118 to activation by TGF-beta, which varies between tissues, ultimately determines the strength of the TGF-beta effect on BGN.     

Synthesis and biological evaluation of 1-substituted-3(5)-(6-methylpyridin-2-yl)-4-(quinolin-6-yl)pyrazoles as transforming growth factor-β type 1 receptor kinase inhibitors

A series of 1-substituted-3(5)-(6-methylpyridin-2-yl)-4-(quinolin-6-yl)pyrazoles 14a-e, 15a-e, 17a-c, and 18a-d have been synthesized and evaluated for their ALK5 inhibitory activity in an enzyme assay and in a cell-based luciferase reporter assay. The 6-quinolinyl pyrazole analogue 14b inhibited ALK5 phosphorylation with IC(50) value of 0.022 μM and showed 84% inhibition at 0.1 μM in a luciferase reporter assay using HaCaT cells permanently transfected with p3TP-luc reporter construct.