SB525334

SB525334 is a potent inhibitor of transforming growth factor-beta1 (TGF-beta1) receptor, activin receptor-like kinase (ALK5). It specifically inhibits ALK5 with IC50 value of 14.3nM, which shows 4-fold higher potency against ALK4 [1].

SB525334 has been demonstrated to reduce smad2/3 nuclear fluorescence induced by TGF-beta1 in human renal proximal tubule epithelial (RPTE) cells. Additionally, SB525334 inhibits TGF-beta1 mediated procollagen and PAI-1 expression in human renal carcinoma cells A498 [1].

SB525334?dose-dependently reduces Urinary protein, procollagen alpha1(I) mRNA and?procollagen alpha1(III) mRNA when administered?orally in acute puromycin aminonucleoside (PAN) rat model of renal disease [1].

References:
[1] Grygielko ET1,?Martin WM,?Tweed C,?Thornton P,?Harling J,?Brooks DP,?Laping NJ. Inhibition of gene markers of fibrosis with a novel inhibitor of transforming growth factor-beta type I receptor kinase in puromycin-induced nephritis. J Pharmacol Exp Ther.?2005 Jun;313(3):943-51.

Follistatin-like 1 protects cardiomyoblasts from injury induced by sodium nitroprusside through modulating Akt and Smad1/5/9 signaling.[Pubmed:26687945]

Biochem Biophys Res Commun. 2016 Jan 15;469(3):418-23.

Cardiac cell apoptosis provoked by excessive sodium nitroprusside (SNP) toxicity, a potent vasodilator, limited its clinical application. Effective means for protection against SNP-induced cardiotoxicity would be highly needed. This study investigated the effects of Follistatin-like 1 (FSTL1) on the injury induced by SNP in rat cardiomyoblast H9c2 cells. First, expression of FSTL is attenuated following SNP treatment. SNP challenge significantly increases cardiac cell death, which is attenuated by FSTL1 pretreatment. Additionally, knockdown of endogenous FSTL1 enhances SNP-induced cell apoptosis. Furthermore, FSTL1 pretreatment partially inhibits SNP-induced NO generation. LY294002 and BMP4 completely abolish cytoprotective role of FSTL1 against SNP challenge, indicating both activation of Akt and inhibition of BMP/Smad1/5/9 signaling are involved in this cellular process. Lastly, FSTL1-mediated cytoprotection is independent of Smad2/3 signaling, as SB525334 fails to remove its protective role. Taken together, these results indicated that FSTL1 protects the SNP-induced injury in cardiac H9c2 cells through, at least in part, the activation of Akt and inhibition of Smad1/5/9 signaling.

Ginsenoside Rg1 Ameliorates Cigarette Smoke-Induced Airway Fibrosis by Suppressing the TGF-beta1/Smad Pathway In Vivo and In Vitro.[Pubmed:28421197]

Biomed Res Int. 2017;2017:6510198.

Small airway fibrosis is a key pathological process accompanying chronic obstructive pulmonary disease (COPD) and includes fibroblast/myofibroblast transdifferentiation and excessive extracellular matrix deposition. Ginsenoside Rg1, one of the main active ingredients of Panax ginseng, has been shown to exert an antifibrotic effect in many tissues. However, little is known about the underlying mechanism and whether ginsenoside Rg1 can exert an effect on small airway fibrosis. We investigated the anti-small airway fibrosis effects of ginsenoside Rg1 in human embryonic lung fibroblasts and in COPD rats. We found that ginsenoside Rg1 effectively reduced the degree of pulmonary fibrosis, decreased the expression of alpha-smooth muscle actin, collagen I, and matrix metalloproteinase 9, and maintained the ratio of matrix metalloproteinase 9 to tissue inhibitor of metalloproteinase 1. Importantly, ginsenoside Rg1 significantly attenuated cigarette smoke extract-induced upregulation of transforming growth factor beta1, TGF-beta receptor I, phospho-Smad2, and phospho-Smad3. In addition, ginsenoside Rg1 mimicked the effect of SB525334, a TGF-beta receptor I-Smad2/3 inhibitor. Collectively, these results suggest that ginsenoside Rg1 may suppress cigarette smoke-induced airway fibrosis in pulmonary fibroblasts and COPD rats by inhibiting the TGF-beta1/Smad signaling pathway.

mTOR inhibition by rapamycin increases ceramide synthesis by promoting transforming growth factor-beta1/Smad signaling in the skin.[Pubmed:27239444]

FEBS Open Bio. 2016 Feb 24;6(4):317-25.

Although mammalian target of rapamycin (mTOR) mediates a wide variety of biological functions, little information is available on the effect of mTOR on the functions of skin cells. In this study, we investigated effects of mTOR inhibition by rapamycin on ceramide synthesis in the skin of rats and human keratinocytes and its regulatory mechanisms. The phosphorylation of p70 S6 kinase, which indicates mTOR activation, was induced in the skin of rats fed a high-fat diet, but this abnormality was reversed by supplementation with rapamycin. Ceramide levels and the mRNA levels of serine palmitoyltransferase (SPT) and transforming growth factor (TGF)-beta1 were suppressed in the skin of rats fed high-fat diets, but this abnormality was reversed by supplementation with rapamycin. TGF-beta1-induced SPT mRNA expression was blocked by SB525334, an inhibitor of TGF-beta1-induced Smad2/3 nuclear localization, in human keratinocytes. Rapamycin-induced SPT mRNA expression was blocked by an anti-TGF-beta1 antibody or SB525334 in human keratinocytes. These results show that mTOR inhibition by rapamycin increases ceramide synthesis by promoting TGF-beta1/Smad signaling in the skin.

The effect of Echinococcus granulosus on spleen cells and TGF-beta expression in the peripheral blood of BALB/c mice.[Pubmed:28130828]

Parasite Immunol. 2017 Mar;39(3).

Cystic echinococcosis (CE) caused by the cestode Echinococcus granulosus (E. granulosus) is a zoonotic parasitic disease. The effective immune evasion mechanisms of E. granulosus allow it to parasitize its hosts. However, the status of the innate and adaptive immune cells and their contributions to E. granulosus progression remain poorly understood. In this study, we aimed to determine the impact of E. granulosus infection on T cells, NK cell responses and TGF-beta expression during the early infection phase in BALB/c mice. In E. granulosus infections, there was an increasing tendency in the percentage of CD4(+) CD25(+) T cells and CD4(+) Foxp3(+) T cells and peripheral blood TGF-beta levels and relative expression of the Foxp3 gene. Moreover, there were a decreasing tendency in the percentage of NK cells and NK cell cytotoxicity and the expression of NKG2D on NK cells. The TGF-beta1/Smad pathway was activated by E. granulosus in mice. Above results can be reversed by the inhibitor SB-525334 (potent activin receptor-like kinase 5 inhibitor). These results suggest that the TGF-beta/Smad pathway plays an important role in changes of T-cell or NK cell responses. These results may contribute to revealing the preliminary molecular mechanisms in establishing hydatid infection.

Apocynin inhibits the upregulation of TGF-beta1 expression and ROS production induced by TGF-beta in skeletal muscle cells.[Pubmed:26321737]

Phytomedicine. 2015 Sep 15;22(10):885-93.

BACKGROUND: Pure apocynin, which can be traditionally isolated and purified from several plant species such as Picrorhiza kurroa Royle ex Benth (Scrophulariaceae), acts as an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activity inhibiting its production of reactive oxygen species (ROS). Transforming growth factor type beta 1 (TGF-beta1) is a growth factor that produces inhibition of myogenesis, diminution of regeneration and induction of atrophy in skeletal muscle. The typical signalling that is activated by TGF-beta involves the Smad pathway. PURPOSE: To evaluate the effect of TGF-beta and the effect of apocynin on TGF-beta1 expression in skeletal muscle cells. STUDY DESIGN: Controlled laboratory study. In vitro assays were performed with C2C12 cells incubated with TGF-beta1 in presence or absence of apocynin (NOX inhibitor), SB525334 (TGF-beta-receptor I inhibitor), or chelerythrine (PKC inhibitor). METHODS: TGF-beta1 and atrogin-1 expression was evaluated by RT-qPCR and/or ELISA; Smad3 phosphorylation by western blot; Smad4 nuclear translocation by indirect immunofluorescence; and ROS levels by DCF probe fluorescent measurements. RESULTS: We show that myoblasts respond to TGF-beta1 by increasing its own gene expression in a time- and dose-dependent fashion which was abolished by SB525334 and siRNA for Smad2/3. TGF-beta1 also induced ROS. Remarkably, apocynin inhibited the TGF-beta1 induced ROS as well as the autoinduction of TGF-beta1 gene expression. We also show that TGF-beta-induced ROS production and TGF-beta1 expression require PKC activity as indicated by the inhibition using chelerythrine. CONCLUSION: These results strongly suggest that TGF-beta induces its own expression through a TGF-beta-receptor/Smad-dependent mechanism and apocynin is able to inhibit this process, suggesting that requires NOX-induced ROS in skeletal muscle cells.

Inhibition of activin receptor-like kinase 5 attenuates bleomycin-induced pulmonary fibrosis.[Pubmed:17274978]

Exp Mol Pathol. 2007 Aug;83(1):39-46.

Activin receptor-like kinase 5 (ALK5) is a type I receptor of transforming growth factor (TGF)-beta. ALK5 inhibition has been reported to attenuate the tissue fibrosis including pulmonary fibrosis, renal fibrosis and liver fibrosis. To elucidate the inhibitory mechanism of ALK5 inhibitor on pulmonary fibrosis in vivo, we performed the histopathological assessment, gene expression analysis of extracellular matrix (ECM) genes and immunohistochemistry including receptor-activated Smads (R-Smads; Smad2/3), CTGF, myofibroblast marker (alpha-smooth muscle actin; aSMA) and type I collagen deposition in the lung using Bleomycin (BLM)-induced pulmonary fibrosis model. ALK5 inhibitor, SB-525334 (10 mg/kg or 30 mg/kg) was orally administered at twice a day. Lungs were isolated 5, 7, 9 and 14 days after BLM treatment. BLM treatment led to significant pulmonary fibrotic changes accompanied by significant upregulation of ECM mRNA expressions, Smad2/3 nuclear translocation, CTGF expression, myofibroblast proliferation and type I collagen deposition. SB-525334 treatment attenuated the histopathological alterations in the lung, and significantly decreased the type I and III procollagen and fibronectin mRNA expression. Immunohistochemistry revealed that SB-525334 treatment showed significant attenuation in Smad2/3 nuclear translocation, decrease in CTGF-expressing cells, myofibroblast proliferation and type I collagen deposition. These results suggest that ALK5 inhibition attenuates R-Smads activation thereby attenuates pulmonary fibrosis.

Tumor-specific efficacy of transforming growth factor-beta RI inhibition in Eker rats.[Pubmed:17505012]

Clin Cancer Res. 2007 May 15;13(10):3087-99.

PURPOSE: Transforming growth factor beta (TGF-beta), which generally stimulates the growth of mesenchymally derived cells but inhibits the growth of epithelial cells, has been proposed as a possible target for cancer therapy. However, concerns have been raised that whereas inhibition of TGF-beta signaling could be efficacious for lesions in which TGF-beta promotes tumor development and/or progression, systemic pharmacologic blockade of this signaling pathway could also promote the growth of epithelial lesions. EXPERIMENTAL DESIGN: We examined the effect of a TGF-beta inhibitor on mesenchymal (leiomyoma) and epithelial (renal cell carcinoma) tumors in Eker rats, which are genetically predisposed to develop these tumors with a high frequency. RESULTS: Blockade of TGF-beta signaling with the ALK5/type I TGF-beta R kinase inhibitor, SB-525334, was efficacious for uterine leiomyoma; significantly decreasing tumor incidence and multiplicity, and reducing the size of these mesenchymal tumors. However, SB-525334 was also mitogenic and antiapoptotic for epithelial cells in the kidney and exacerbated the growth of epithelial lesions present in the kidneys of these animals. CONCLUSION: Although pharmacologic inhibition of TGF-beta signaling with SB-525334 may be efficacious for mesenchymal tumors, inhibition of this signaling pathway seems to promote the development of epithelial tumors.

Inhibition of gene markers of fibrosis with a novel inhibitor of transforming growth factor-beta type I receptor kinase in puromycin-induced nephritis.[Pubmed:15769863]

J Pharmacol Exp Ther. 2005 Jun;313(3):943-51.

SB-525334 (6-[2-tert-butyl-5-(6-methyl-pyridin-2-yl)-1H-imidazol-4-yl]-quinoxaline) has been characterized as a potent and selective inhibitor of the transforming growth factor-beta1 (TGF-beta1) receptor, activin receptor-like kinase (ALK5). The compound inhibited ALK5 kinase activity with an IC(50) of 14.3 nM and was approximately 4-fold less potent as an inhibitor of ALK4 (IC(50) = 58.5 nM). SB-525334 was inactive as an inhibitor of ALK2, ALK3, and ALK6 (IC(50) > 10,000 nM). In cell-based assays, SB-525334 (1 microM) blocked TGF-beta1-induced phosphorylation and nuclear translocation of Smad2/3 in renal proximal tubule cells and inhibited TGF-beta1-induced increases in plasminogen activator inhibitor-1 (PAI-1) and procollagen alpha1(I) mRNA expression in A498 renal epithelial carcinoma cells. In view of this profile, SB-525334 was used to investigate the role of TGF-beta1 in the acute puromycin aminonucleoside (PAN) rat model of renal disease, a model of nephritis-induced renal fibrosis. Orally administered doses of 1, 3, or 10 mg/kg/day SB-525334 for 11 days produced statistically significant reductions in renal PAI-1 mRNA. Also, the compound produced dose-dependent decreases in renal procollagen alpha1(I) and procollagen alpha1(III) mRNA, which reached statistical significance at the 10-mg/kg/day dose when compared with vehicle-treated PAN controls. Furthermore, PAN-induced proteinuria was significantly inhibited at the 10-mg/kg/day dose level. These results provide further evidence for the involvement of TGF-beta1 in the profibrotic changes that occur in the PAN model and for the first time, demonstrate the ability of a small molecule inhibitor of ALK5 to block several of the markers that are predictive of fibrosis and renal injury in this model.