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Filter-binding assay
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The IC50 values of LY364947 at different enzyme concentrations were determined by the filter-binding assay. Typically, 40 μL reactions in 50 mM HEPES at pH 7.5, 1 mM NaF, 200 μM pKSmad3(-3) and 50 mM ATP containing a titration of each inhibitor with concentrations of 1600, 800, 400, 200, 100, 50, 25, and 0 nM were incubated at 30 °C for 30 mins. The IC50 values were calculated using a nonlinear regression method with GraphPad Prism software. The binding type was determined by plotting the correlation between enzyme concentrations and IC50 values.
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Applications
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In HOXB9-MCF10A cells, LY364947 suppressed Smad2 phosphorylation by inhibiting TGF-β activation, meanwhile, without affecting the expression of TGF-β1 and TGF-β2. Besides, LY364947 induced epithelial morphological changes, with re-expression of E-cadherin as well as suppression of fibronectin and vimentin. In addition, LY364947 reduced migration and invasiveness of HOXB9-MCF10A cells.
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Applications
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In a rat model of NMDA-induced retinal degeneration, LY364947 significantly suppressed cell loss in the ganglion cell layer induced by NMDA. Besides, LY364947 markedly prevent vascular damage in the injured retina caused by NMDA. In addition, co-treatment with NMDA and LY364947 did not cause any morphological change of NG2-positive pericytes.
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References:
[1]. Peng SB, Yan L, Xia X, Watkins SA, Brooks HB, Beight D, Herron DK, Jones ML, Lampe JW, McMillen WT, Mort N, Sawyer JS, Yingling JM. Kinetic characterization of novel pyrazole TGF-beta receptor I kinase inhibitors and their blockade of the epithelial-mesenchymal transition. Biochemistry. 2005 Feb 22;44(7):2293-304.
[2]. Hayashida T, Takahashi F, Chiba N, Brachtel E, Takahashi M, Godin-Heymann N, Gross KW, Vivanco Md, Wijendran V, Shioda T, Sgroi D, Donahoe PK, Maheswaran S. HOXB9, a gene overexpressed in breast cancer, promotes tumorigenicity and lung metastasis. Proc Natl Acad Sci U S A. 2010 Jan 19;107(3):1100-5.
[3]. Ueda K, Nakahara T, Mori A, Sakamoto K, Ishii K. Protective effects of TGF-β inhibitors in a rat model of NMDA-induced retinal degeneration. Eur J Pharmacol. 2013 Jan 15;699(1-3):188-93.
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