We propose to develop and configure for high throughput screening an assay that targets the key protein- protein interaction required for transcriptional responses to transforming growth factor beta (TGF-beta). The protein-protein interaction targeted by the assay is the binding between the regulated Smad (R-Smad) transcription factors and Smad4. There are currently no small molecule ligands that target Smad-Smad binding. Inhibitors of this protein interaction would be useful in characterizing the myriad of biological responses to TGF-beta in different cell types and could be used as lead compounds to develop pharmacological inhibitors of TGF-beta signaling. Small molecule inhibition at the level of the Smads might have a more selective effect on cellular responses to TGF-beta than inhibition of the ligand or receptor. Inhibition of TGF- beta signaling is a validated target in several advanced cancers because of its role in facilitating cancer cell migration, proliferation, metastasis to bone and evasion of the immune response. It is also a validated target in all forms of fibrotic disease including idiopathic pulmonary fibrosis and diabetic nephropathy.
The Specific Aims i nclude development of protein reagents and a homogenous time-resolved fluorescence resonance energy transfer readout, optimization of parameters and binding specificity and reversibility in 384-well format, statistical evaluation of the assay, development of secondary assays and counter screens and implementation in a pilot screen of an available library of 4000 bioactive compounds. If the Aims of this application are achieved, future implementation of the assay at UW and the MLSCN should identify novel compounds targeted to the Smad-Smad interface, a protein-protein interaction with known molecular (co- crystal) structure. The compounds would provide critical research reagents to demonstrate in pre-clinical animal models which normal or pathological responses to TGF-beta depend on the R-Smad-Smad4 interaction. Given the need for new therapeutic approaches in diseases such as glioma and idiopathic pulmonary fibrosis, where TGF- beta signaling is an important target, active compounds identified by the proposed assay would have a high probability of stimulating additional pharmaceutical development and clinical trials.
