The TGF-β isoforms, TGF-β1, -β2, and –β3, are well-known to promote the progression of several different soft tissue cancers, such as those of the breast, brain, prostate, liver, and lung, as well as promote the accumulation of extracellular matrix (ECM) that leads to the progression of fibrotic disorders, such as idiopathic pulmonary fibrosis, cardiac fibrosis, and renal fibrosis. The therapeutic benefit of antagonizing TGF- βs using small molecule receptor kinase inhibitors (SMRKIs), neutralizing antibodies (NABs), and other approaches has been amply demonstrated in animals, yet no inhibitors have been approved for treatment of cancer or fibrosis in humans. The SMRKIs have poor specificity/selectivity and have failed in clinical trials. Biologics, such as TGF-β pan-isoform NABs, are highly specific and safe, but penetrate poorly into dense tissues such as tumors and may be unable to effectively bind and neutralize TGF-βs, which are stored in the extracellular matrix (ECM) as a latent protein bound to their pro-domain, and indirectly, to other ECM proteins such as LTBP and GARP. The objective of this proposal is to leverage the high specificity of the TGF-βs for their type II receptor, TβRII, as well as our understanding of the underlying structural basis for this specificity, to discover and develop a novel class of small molecule assembly inhibitors (SMAIs) that bind either to the fingertip region of TGF-β or to the corresponding interacting surface of TβRII to block TGF-β:TβRII binding and the subsequent recruitment of TbRI and signaling. The hypothesis of our proposed research is that SMAIs that bind in this manner should effectively target the TGF-β pathway in a highly specific manner. This, together with increased accessibility of an extracellular target for the SMAIs, rather than an intracellular target for the SMRKIs, should increase the effectiveness of the SMAIs. To discover and develop this promising new class of small molecule TGF-β inhibitors, we will employ unique protein reagents, developed over many years in one of the PI’s laboratory, that will enable the reliable identification of inhibitors using a highly sensitive TR- FRET high throughput screening (HTS) assay that we have developed, optimized, and validated. To enable the reliable identification of bona fide inhibitors, we will employ two counter screens, a TR-FRET interference and an orthogonal assay format. We will utilize a panel of cell-based assays to assess pathway selectivity, potency, and interference with TGF-β stimulated activities, such as EMT and deposition of ECM, that are known to drive disease progression. To enable future optimization of a lead compound, we will identify the target protein and determine the structure of the inhibitor bound to the target protein using X-ray crystallography or NMR and develop an initial SAR based on evaluation of available analogs in biophysical and functional assays.