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

Smad 依赖性 TGF-be 转录反应抑制剂的 HTS 测定

• 批准号: 7169724
• 负责人: F. Michael Hoffmann
• 金额: $ 18.1万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7169724
• 关键词: NIH Roadmap Initiative tag; chemical registry /resource; drug discovery /isolation; fluorescence resonance energy transfer; genetic transcription; high throughput technology; inhibitor /antagonist; pharmacology; protein binding; protein protein interaction; protein structure function; small molecule; technology /technique development; transcription factor; transforming growth factors

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.

描述（由申请人提供）：我们建议开发和配置一种用于高通量筛选的测定法，该测定法针对转化生长因子β（TGF-β）的转录反应所需的关键蛋白质-蛋白质相互作用。该检测所针对的蛋白质-蛋白质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。这种蛋白质相互作用的抑制剂将有助于表征不同细胞类型中对 TGF-β 的多种生物反应，并且可以用作先导化合物来开发 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抑制配体或受体对细胞对 TGF-β 的反应具有更具选择性的影响。抑制 TGF-β 信号传导是多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方面发挥着作用。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同质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、测定的统计评估、二次测定和计数器筛选的开发以及实施对 4000 种生物活性化合物的可用库进行试点筛选。如果该应用的目标得以实现，未来在威斯康辛大学和 MLSCN 实施的检测应能鉴定出针对 Smad-Smad 界面的新化合物，该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，以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 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，其中TGF-β信号传导是一个重要靶点，通过所提出的测定法鉴定出的活性化合物将很有可能刺激更多的药物开发和临床试验。