Targeting a protein interaction site on Smad transcription factors
Principal Investigator/Assay Provider:
Michael Hoffman, PhD, University of Wisconsin
dentify small molecule inhibitors of Smad function
Smad transcription factors mediate cellular responses to transforming growth factor beta (TGF-b). Inhibition of Smad function will be an important research tool in elucidating the multiple biological functions of TGF-b. TGF-b signaling is a validated target in several advanced cancers because of its role in facilitating cancer cell migration, proliferation, metastasis to bone and/or evasion of the immune response. It is also a validated target in all forms of fibrotic disease including idiopathic pulmonary fibrosis and diabetic nephropathy. Current strategies for inhibiting TGF-b signaling focus on disruption of the ligand-receptor interaction with neutralizing antibodies or inhibition of the TGF-b receptors using small molecule inhibitors of the kinase activity. There are no small molecule ligands that target Smad proteins. This project targets the transcriptionally active Smad complexes to inhibit TGF-b signaling. The working hypothesis is that disruption of only one binding site on Smad will block binding of some but not all Smad binding partners (over two dozen different transcription factors, coactivators and co-repressors), thereby interfering with only a subset of the gene expression responses mediated by Smad complexes. One advantage of this strategy over inhibiting ligand binding or receptor kinase activity is that it might provide selective inhibition of some but not all TGF-b responses. If the aims of this application are achieved, new compounds targeted to the Smad protein hydrophobic corridor would become critical research reagents to demonstrate which normal or pathological responses to TGF-b depend on this Smad protein binding site. Given the need for new therapeutic approaches in diseases where TGF-b signaling is an important target, such as glioma and idiopathic pulmonary fibrosis, active compounds identified by the proposed assay would stimulate additional pharmaceutical development and clinical trials.
This assay is a homogeneous time-resolved fluorescence resonance transfer (HTR-FRET) assay that measures peptide binding to a specific protein binding site in the hydrophobic corridor of Smad2 and Smad3. The binding site is present only in Smad2 and Smad3 and not in Smads 1, 4, 5, 6, 7, or 8, therefore, ligands to this site should have preferential effects on Smad2 and Smad3 functions. For screening, the binding of a the 24-amino acid FoxH1 Smad interaction motif (SIM) to purified, full-length, His-tagged, phosphorylated Smad3 in the presence or absence of library compounds is determined by measuring FRET between a europium-anti-6XHis antibody bound to Smad3 (FRET donor) and the Dy-647 label on the SIM (FRET acceptor).