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

众所周知，TGF-β同种型TGF-β1、TGF-β2和TGF-β3可促进几种不同肿瘤的进展。 软组织癌，如乳腺癌、脑癌、前列腺癌、肝癌和肺癌，以及促进 细胞外基质（ECM）的积累，导致纤维化疾病的进展，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 使用小分子受体激酶抑制剂（SMRKI）、中和抗体（NAB）和其他 这些方法已经在动物中得到了充分的证明，但还没有抑制剂被批准用于治疗 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，如TGF-β泛亚型NAB，具有高度特异性和安全性，但渗透到致密组织中的能力较差。 组织如肿瘤，并且可能不能有效地结合和中和TGF-βs，TGF-βs储存在组织中。 细胞外基质（ECM）作为一种潜在的蛋白质结合到他们的前结构域，并间接地，其他ECM蛋白 例如LTBP和GARP。该提案的目的是利用TGF-β的高度特异性， 它们的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， 发现和开发一类新的小分子组装抑制剂（SMAI）， 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的募集和信号传导。我们提出的研究假设是， 以这种方式结合的细胞应该以高度特异性的方式有效地靶向TGF-β途径。连同 SMAI的细胞外靶点的可及性增加，而不是SMAI的细胞内靶点。 SMRKIs应该增加SMAIs的有效性。去发现和发展这个有前途的新阶级 小分子TGF-β抑制剂，我们将采用独特的蛋白质试剂，多年来开发， PI的实验室之一，这将使可靠的识别抑制剂使用高度敏感的TR- FRET高通量筛选（HTS）分析，我们已经开发，优化和验证。使 为了可靠地鉴定真正的抑制剂，我们将采用两种计数器筛选，一种TR-FRET干扰， 和正交测定形式。我们将利用一组基于细胞的测定来评估途径选择性， 效力，以及干扰TGF-β刺激的活性，如EMT和ECM沉积， 已知会导致疾病进展。为了使先导化合物的未来优化成为可能，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，并根据生物物理学中可用类似物的评价开发初始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.