Targeting the Sam68-stimulated PARP1 activation for cancer treatment

项目摘要 抑制聚(ADP-核糖)聚合酶1(PARP1)蛋白已成为一种很有前途的研究方向 几种癌症的治疗方法。然而，目前的PARP1抑制剂类别都是 烟酰胺的衍生物，基于与烟酰胺竞争结合的策略。因为 PARP1催化的PAR化在许多细胞过程中是关键的，PARP1的治疗靶点在 含有当前类别的PARP1抑制剂的癌细胞可能会对正常的许多细胞功能产生负面影响 细胞。临床上迫切需要提高PARP1抑制剂的特异性，降低Off-TdR。 靶向效应和毒性，以获得更好的治疗价值和方式。然而，快速招聘的 DNA损伤部位的PARP1(毫秒内)和强烈合成的PAR链使其 很难阐明PARP1激活的确切刺激和调节机制。我们最近 发现Src-associated-substrate-during-mitosis-of-68kDa(Sam68)在 刺激DNA损伤特异性PARP1的激活，这表明针对Sam68刺激的 PARP1激活可能成为开发新型PARP1抑制剂的一种新策略。的目标是 我们的研究是通过高密度脂蛋白识别Sam68刺激的PARP1激活的特异性分子抑制剂 通过筛选，我们的假设是这些抑制剂将减轻DNA损伤引发的 因此，PARP1的激活可能成为治疗癌症的潜在药理药物。我们已经开发出一种 适用于小分子文库筛选的新型酶联免疫吸附试验。在这项拟议的研究中，我们将 进行高通量筛选以鉴定抑制Sam68刺激的小分子化合物 PARP1激活。我们将采用跨学科的方法，结合生化、生物物理学和 分子实验、细胞分析和成像方法，以验证筛选命中和识别 高质量的Sam68-PARP1相互作用小分子抑制剂。最有效的化合物的活性将会 在一组基于细胞的和小鼠模型实验中进行评估，以评估它们抑制 结肠癌细胞的存活和发展。我们的项目代表了一种非常创新的战略，以抑制 PARP1活性通过阻断Sam68赋予的刺激机制而发挥作用。在这些结束时 在研究中，我们希望能够识别出具有很高价值的化合物，这些化合物可以作为化学探针，适用于 进一步开发用于癌症治疗的有效的PARP1抑制剂。此外，这些发现 抑制剂将极大地促进我们对Sam68刺激的PARP1的调节和功能的研究 在正常和疾病条件下激活。

Project Summary The inhibition of poly(ADP-ribose) polymerase 1 (PARP1) protein has emerged as a promising therapeutic treatment for several types of cancers. However, the current classes of PARP1 inhibitors are all derivatives of nicotinamide, based on the strategy of competitive binding against nicotinamide. Because the PARP1-catalyzed PARylation is critical for many cellular processes, therapeutic targeting of PARP1 in cancerous cells with current classes of PARP1 inhibitors could negatively affect many cell functions in normal cells. There is an urgent clinical need for improving the inhibitor specificity for PARP1 and lowering the off- target effects and toxicity for better therapeutic value and modalities. However, the rapid recruitment of PARP1 to sites of DNA lesions (within milliseconds) and the vigorously synthesized PAR chains make it difficult to elucidate the precise stimulatory and regulatory mechanisms on PARP1 activation. We recently discovered that Src-associated-substrate-during-mitosis-of-68kDa (Sam68) plays an important role in stimulating the DNA damage-specific PARP1 activation, which suggests that targeting the Sam68-stimulated PARP1 activation could be a novel strategy to develop a new category of PARP1 inhibitors. The objective of our research is to identify specific molecule inhibitors of the Sam68-stimulated PARP1 activation via a high throughput screening, and our hypothesis is that these inhibitors would alleviate DNA damage-triggered PARP1 activation thus being potential pharmacological agents for treating cancers. We have developed a novel ELISA-based assay suitable for screening of small molecule libraries. In this proposed research, we will conduct high throughput screening to identify small molecule compounds inhibiting the Sam68-stimulated PARP1 activation. We will employ interdisciplinary approach combining biochemical, biophysical, and molecular experiments, cellular assays, and imaging methods to validate the screening hits and to identify high quality small molecule inhibitors of Sam68-PARP1 interaction. Activity of the most potent compounds will be evaluated in a panel of cell-based and mouse model experiments to assess their capability to inhibit the survival and development of colon cancer cells. Our project represents a very innovative strategy to inhibit PARP1 activity through blocking the Sam68-conferred stimulatory mechanism. At the conclusion of these studies, we expect to identify highly valuable compounds that can serve as chemical probes suitable for further development into potent PARP1 inhibitors for cancer therapeutics. Additionally, discovery of these inhibitors will greatly facilitate our research on the regulation and function of the Sam68-stimulated PARP1 activation under normal and disease conditions.