| Description |
ITD-1 is the first selective TGFβ inhibitor with an IC50 of 460 nM.
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| Related Catalog |
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| Target |
IC50: 460 nM (TGFβ)
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| In Vitro |
ITD-1 potently blocks phosphorylation of the effector SMAD2/3 proteins induced by TGFβ2, and only minimally in response to Activin A. HEK293T cells are transfected with a Smad4 response element driving luciferase (SBE4-Luc) to test whether ITD-1 blocks Activin A/Nodal and/or TGFβ signaling, which utilize the same intracellular signaling cascade through Smad4. ITD-1 strongly inhibits TGFβ2 signaling with similar efficacy (92% vs. 99% respectively), but with lower potency compared to SB-431542, an ACVR1B/TGFBR1 kinase inhibitor (IC50= 850nM vs. 70nM respectively), and is a weak and partial inhibitor of Activin A signals. ITD-1 selectively enhances the differentiation of uncommitted mesoderm to cardiomyocytes, but not to vascular smooth muscle and endothelial cells. ITD-1 reveals an unexpected role for TGFβ signaling in controlling cardiomyocyte differentiation from multipotent cardiovascular precursors.
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| Cell Assay |
For the Smad4-Response element (SBE4) assay, 293T cells are grown in Phenol Free DMEM-high glucose supplemented with 1% FBS. About 30000 cells/cm2 are reverse transfected onto white 384-well cell-culture plates with 10ng of SBE4-Lux and CMV-Renilla-Lux using Lipofectamine 2000. Cells are allowed to adhere for at least 12 hours and induced with either TGFβ2 (15 ng/mL), Activin A (15 ng/mL). Simultaneously, ITD-1 (0.001, 0.01, 0.1, 1, and 10 μM) is added to cells. SB-431542 is used as a positive control for Activin A/TGFβ-signaling inhibition. To determine luminescence levels, Dual-Glo kit is used and measured on an Envision plate reader. Firefly luminescence is normalized against renilla luciferase.
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| References |
[1]. Willems E, et al. Small molecule-mediated TGF-β type II receptor degradation promotes cardiomyogenesis in embryonic stem cells. Cell Stem Cell. 2012 Aug 3;11(2):242-52.
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