An HTS Assay for Inhibitors of Smad-dependent Transcriptional Responses to TGF-be

描述（由申请人提供）：我们建议开发和配置用于高通量筛选的检测，该检测针对转化生长因子β (tgf - β)转录反应所需的关键蛋白质-蛋白质相互作用。该实验针对的蛋白-蛋白相互作用是受调节的Smad （R-Smad）转录因子与Smad4之间的结合。目前还没有针对Smad-Smad结合的小分子配体。这种蛋白相互作用的抑制剂将有助于表征不同细胞类型中对tgf - β的无数生物反应，并可作为先导化合物开发tgf - β信号传导的药理学抑制剂。Smads水平的小分子抑制可能比配体或受体的抑制对细胞对tgf - β的反应有更多的选择性作用。TGF- β信号的抑制是几种晚期癌症的有效靶点，因为它在促进癌细胞迁移、增殖、骨转移和逃避免疫反应中发挥作用。它也是所有形式的纤维化疾病，包括特发性肺纤维化和糖尿病肾病的有效靶点。具体目标包括开发蛋白质试剂和均匀的时间分辨荧光共振能量转移读数，优化384孔格式的参数和结合特异性和可逆性，统计分析评估，开发二次分析和计数器筛选，并在4000个生物活性化合物的可用文库中进行试点筛选。如果这项应用的目的得以实现，未来在华盛顿大学和MLSCN的实验实施应该识别针对Smad-Smad界面的新化合物，Smad-Smad界面是一种具有已知分子（共晶）结构的蛋白质-蛋白质相互作用。这些化合物将提供关键的研究试剂，在临床前动物模型中证明对tgf - β的正常或病理反应取决于R-Smad-Smad4的相互作用。鉴于神经胶质瘤和特发性肺纤维化等疾病需要新的治疗方法，TGF- β信号是这些疾病的重要靶点，因此通过所提出的检测确定的活性化合物将极有可能刺激额外的药物开发和临床试验。

DESCRIPTION (provided by applicant): 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 include 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.