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

众所周知，转化生长因子-β亚型，转化生长因子-β1、-β2和-β3，可促进多种不同的肿瘤进展。 软组织癌症，如乳腺癌、脑癌、前列腺癌、肝癌和肺癌，并促进 细胞外基质(ECM)的积聚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。拮抗转化生长因子-α的治疗作用 βS使用小分子受体激酶抑制剂(SMRKI)、中和抗体(NAB)等 这种方法已经在动物身上得到了充分的证明，但还没有抑制剂被批准用于治疗 人类的癌症或纤维化。SMRKI的特异性/选择性较差，临床试验失败。 生物制剂，如转化生长因子-β泛异构体NaB，具有高度的特异性和安全性，但对稠密的渗透较差。 肿瘤等组织，可能无法有效结合和中和储存在体内的转化生长因子-βS 细胞外基质(ECM)作为一种潜伏蛋白与其原结构域结合，并间接与其他ECM蛋白结合 例如LTBP和GARP。这项建议的目的是利用转化生长因子-β的高度特异性为S 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， 为了发现和开发一类新型的小分子组装抑制剂(SMAI)，它可以结合到 转化生长因子-β的指尖区域或相应的T-βRII相互作用表面阻断转化生长因子-β：T-βRII的结合 以及随后的TBRI和信令的招募。我们提议的研究的假设是SMAI 这种结合方式应该以高度特异性的方式有效地靶向转化生长因子-β途径。这一次，一起 随着SMAI的细胞外目标而不是细胞内目标的可获得性增加 中小型企业融资机制应提高中小企业融资机制的效力。发现和发展这一有前途的新阶层 在小分子转化生长因子-β抑制剂中，我们将使用经过多年开发的独特蛋白质试剂 PI的实验室之一，这将使使用高灵敏的TR-IR可靠地识别抑制剂成为可能。 我们开发、优化和验证的FRET高通量筛选(HTS)方法。要启用 为了可靠地鉴定真正的抑制剂，我们将采用两个计数器筛选，一个tr-fret干扰 和一种正交化验格式。我们将利用一组基于细胞的分析来评估途径的选择性， 效力，并干扰转化生长因子-β刺激的活动，如细胞外基质的转化和细胞外基质的沉积，这些是 已知会推动疾病的发展。为了能够在将来优化先导化合物，我们将确定 并用X射线确定与靶蛋白结合的抑制剂的结构 结晶学或核磁共振，并根据生物物理中可用类似物的评估开发初始SAR 和功能分析。

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.