Targeting the Sam68-stimulated PARP1 activation for cancer treatment

项目摘要 聚（ADP-核糖）聚合酶1（PARP 1）蛋白的抑制已经成为一种有前途的 对几种类型的癌症进行治疗。然而，目前的PARP 1抑制剂种类都是 烟酰胺的衍生物，基于对烟酰胺的竞争性结合的策略。因为 PARP 1催化的PAR化对于许多细胞过程是至关重要的，PARP 1的治疗靶向作用在细胞内。 具有当前类型的PARP 1抑制剂的癌细胞可以对正常人中的许多细胞功能产生负面影响。 细胞临床上迫切需要提高PARP 1的抑制剂特异性并降低其非特异性。 靶向作用和毒性，以获得更好的治疗价值和方式。然而，快速招聘 PARP 1与DNA损伤位点（毫秒内）的结合， 难以阐明PARP 1激活的精确刺激和调节机制。我们最近 发现Src-相关-底物-有丝分裂期间-68 kDa（Sam 68）在 刺激DNA损伤特异性PARP 1激活，这表明靶向Sam 68刺激的 PARP 1激活可能是开发新型PARP 1抑制剂的新策略。的目标 我们的研究是通过高水平的免疫抑制来鉴定Sam 68刺激的PARP 1激活的特异性分子抑制剂。 通量筛选，我们的假设是，这些抑制剂将减轻DNA损伤触发 PARP 1活化因此是用于治疗癌症的潜在药理学试剂。我们已经开发出一种 新的基于ELISA的测定法，适用于筛选小分子文库。在这项研究中，我们将 进行高通量筛选以鉴定抑制Sam 68刺激的细胞凋亡的小分子化合物。 PARP 1激活。我们将采用跨学科的方法，结合生物化学，生物物理， 分子实验、细胞分析和成像方法，以验证筛选命中并识别 Sam 68-PARP 1相互作用的高质量小分子抑制剂。最有效的化合物的活性将 在一组基于细胞的和小鼠模型实验中进行评价，以评估它们抑制 结肠癌细胞的生存和发展。我们的项目代表了一个非常创新的战略， PARP 1活性通过阻断Sam 68赋予的刺激机制。在这些结束时， 研究中，我们希望能够确定非常有价值的化合物，可以作为化学探针，适用于 进一步开发成用于癌症治疗的有效PARP 1抑制剂。此外，发现这些 抑制剂将极大地促进我们对Sam 68刺激的PARP 1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.