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

众所周知，TGF-β 同工型 TGF-β1、-β2 和 –β3 可促进多种不同疾病的进展。 软组织癌，例如乳腺癌、脑癌、前列腺癌、肝癌和肺癌，并促进 细胞外基质（ECM）的积累导致纤维化疾病的进展，例如 特发性肺纤维化、心脏纤维化和肾纤维化。拮抗 TGF-β 的治疗益处 βs 使用小分子受体激酶抑制剂 (SMRKI)、中和抗体 (NAB) 和其他 方法已在动物身上得到充分证明，但尚未批准任何抑制剂用于治疗 人类癌症或纤维化。 SMRKI的特异性/选择性较差，在临床试验中失败。 生物制剂，例如 TGF-β 泛亚型 NAB，具有高度特异性和安全性，但渗透到致密区域的能力较差 组织，如肿瘤，可能无法有效结合和中和 TGF-β，而 TGF-β 储存在 细胞外基质 (ECM) 作为潜在蛋白与其前结构域结合，并间接与其他 ECM 蛋白结合 例如 LTBP 和 GARP。该提案的目的是利用 TGF-β 的高度特异性 他们的 II 型受体 TβRII，以及我们对这种特异性的潜在结构基础的理解， 发现和开发一类新型小分子组装抑制剂（SMAIs），可与 TGF-β 的指尖区域或 TβRII 的相应相互作用表面以阻断 TGF-β:TβRII 结合 以及随后 TbRI 和信号传导的募集。我们提出的研究假设是 SMAI 以这种方式结合的蛋白应该以高度特异性的方式有效地靶向 TGF-β 途径。这个，一起 增加了 SMAI 的细胞外靶点的可及性，而不是 SMAI 的细胞内靶点 SMRKI 应提高 SMAI 的有效性。发现和开发这个有前途的新类别 对于小分子 TGF-β 抑制剂，我们将采用多年来开发的独特蛋白质试剂 PI 的实验室之一，将能够使用高度灵敏的 TR- 技术可靠地识别抑制剂 我们开发、优化和验证的 FRET 高通量筛选 (HTS) 测定法。启用 为了可靠地识别真正的抑制剂，我们将采用两种计数器筛选，一种是 TR-FRET 干扰 和正交测定格式。我们将利用一组基于细胞的测定来评估途径选择性， 效，并干扰 TGF-β 刺激的活动，例如 EMT 和 ECM 沉积， 已知会推动疾病进展。为了实现先导化合物的未来优化，我们将确定 目标蛋白并使用 X 射线确定与目标蛋白结合的抑制剂的结构 晶体学或核磁共振，并根据生物物理学中可用类似物的评估开发初始 SAR 和功能测定。

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.