HTS for TGF-beta receptor assembly inhibitors with anti-tumor and anti-fibrosis activities

具有抗肿瘤和抗纤维化活性的 TGF-β 受体组装抑制剂的 HTS

• 批准号: 10194415
• 负责人: ANDREW P HINCK
• 金额: $ 47.39万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-10 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10194415
• 关键词: Animals; Attenuated; Binding; Binding Proteins; Binding Sites; Biochemical; Biological; Biological Assay; Biophysics; Biotin; Blood Vessels; Brain; Breast; Calorimetry; Cells; Chemicals; Clinical Trials; Complex; Deposition; Development; Disease; Disease Progression; Effectiveness; Energy Transfer; Evaluation; Extracellular Domain; Extracellular Matrix; Extracellular Matrix Proteins; Family; Fibrosis; Fluorescence Resonance Energy Transfer; Future; Growth; Growth Factor; Human; In Vitro; Laboratories; Lead; Liver; Lung; Malignant Neoplasms; Malignant neoplasm of brain; Malignant neoplasm of prostate; Modeling; Neoplasm Metastasis; Pathway interactions; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phosphotransferases; Property; Prostate; Protein Isoforms; Proteins; Reagent; Reporter; Research; Safety; Series; Signal Pathway; Signal Transduction; Specificity; Structure; Structure-Activity Relationship; Surface; TGF Beta Signaling Pathway; Testing; Therapeutic; Time; Tissues; Titrations; Transforming Growth Factor beta; Transforming Growth Factor beta Receptors; Tumor Promoters; Tumor Tissue; X-Ray Crystallography; analog; base; cancer therapy; cell motility; clinical development; coronary fibrosis; extracellular; high throughput screening; idiopathic pulmonary fibrosis; improved; inhibitor/antagonist; kidney fibrosis; kinase inhibitor; lead series; malignant breast neoplasm; milligram; neutralizing antibody; novel; novel strategies; pharmacophore; receptor; recruit; response; small molecule; small molecule inhibitor; soft tissue; tumor; tumor growth

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.

TGF-β同工型TGF-β1，-β2和–β3是众所周知的，可以促进几种不同的进展 软组织癌，例如乳房，大脑，前列腺，肝脏和肺的癌症，并促进 细胞外基质（ECM）的积累，导致纤维化疾病的发展，例如 特发性肺纤维化，心脏纤维化和肾纤维化。对抗TGF-的治疗益处 使用小分子受体激酶抑制剂（SMRKI），中和抗体（NABS）和其他 动物中已经充分证明了方法，但是尚无抑制剂用于治疗 人类的癌症或纤维化。 SMRKI的特异性/选择性差，并且在临床试验中失败。 生物制剂，例如TGF-β泛氧化型NAB，是高度特异性和安全的，但渗透到密集中 肿瘤等组织，可能无法有效地结合和中和TGF-β，这些TGF-β存储在 细胞外基质（ECM）作为与其亲域的潜在蛋白质，并间接与其他ECM蛋白 例如LTBP和GARP。该提案的目的是利用TGF-β的高特异性 他们的II型受体TβRII，以及我们对这种特异性的基本结构基础的理解， 发现和开发一类新型的小分子组装抑制剂（SMAI），它们与 TGF-β的指尖区域或TβRII相应相互作用的表面以阻断TGF-β：TβRII结合 以及随后的TBRI和信号传导的募集。我们提出的研究的假设是SMAI 以这种方式结合的应该有效地以高度特异性的方式靶向TGF-β途径。这，在一起 随着SMAI的细胞外靶标的可及性提高，而不是针对的细胞内靶 SMRKIS应提高SMAI的有效性。发现和发展这个诺言新班 在小分子TGF-β抑制剂中，我们将采用多年来开发的独特蛋白质试剂 PI的一个实验室之一，它将能够使用高度敏感的TR-可靠地识别抑制剂 FRET高吞吐量筛选（HTS）测定法，我们已经开发，优化和验证了。启用 真正识别真正的FIDE抑制剂，我们将采用两个计数器屏幕，一个TR-FRET干扰 和正交分析格式。我们将利用一组基于细胞的测定法来评估途径选择性， 效力和干扰TGF-β刺激活性（例如EMT和ECM的沉积） 已知可以推动疾病进展。为了实现未来的铅化合物的优化，我们将确定 靶蛋白并使用X射线确定与靶蛋白结合的抑制剂的结构 晶体学或NMR并根据对生物物理中可用类似物的评估开发初始SAR 和功能分析。