Development of Small Molecule Radiation Sensitizers

描述（由申请人提供）： 我们合成了一种新的合成化学文库，并将其用于正向化学遗传学筛选，旨在鉴定内皮形态发生的小分子抑制剂。该筛选导致鉴定了抑制内皮细胞增殖、迁移、在基质胶中形成管状结构的能力以及使用背侧皮肤褶皱血管窗室可视化的新血管生成的结构相关的小分子。生物化学分析显示，新的小分子抑制ENOX 1，一种质膜相关酶，表现出蛋白质二硫键-硫醇交换活性。虽然ENOX活性对细胞增殖很重要，但其分子机制尚未完全了解。shRNA介导的HUVEC中ENOX活性抑制抑制了内皮细胞迁移和基质胶中小管样结构的形成，再现了小分子抑制剂的作用。小分子抑制ENOX 1与电离辐射诱导的内皮细胞凋亡显著增加相关。集落形成试验表明，这些小分子增加了内皮细胞的辐射敏感性。相反，肿瘤上皮细胞的辐射敏感性不受小分子的影响。在分次X射线照射前给予小分子ENOX 1抑制剂，可使表达CD 34的刘易斯肺癌（LLC）肿瘤相关微血管的数量和密度出现统计学显著降低。LLC和人HT 29结肠癌肿瘤模型的使用表明，与单独照射相比，小分子ENOX 1抑制剂与分次X射线照射联用产生肿瘤生长延迟的统计学显著增加。当小鼠接受120 mg/kg推注时，在30天间隔内未观察到急性毒性证据。这些初步的数据支持这一假设，即ENOX 1代表了一个合理的分子靶点，用于增加电离辐射控制肿瘤生长的能力。本申请的短期目标是验证该假设。长期目标是将这些小分子抑制剂开发成具有临床疗效的放射增敏剂。 公共卫生相关性： 正向化学遗传学筛选鉴定内皮形态发生的小分子抑制剂。生物化学分析显示，这些新的小分子抑制酶ENOX 1，增加内皮细胞的辐射敏感性，增加辐射介导的肿瘤相关血管抑制，从而抑制肿瘤生长。这些初步的数据支持这一假设，即ENOX 1代表了一个合理的分子靶点，用于增加电离辐射控制肿瘤生长的能力。

DESCRIPTION (provided by applicant): We synthesized a novel synthetic chemical library and used it in a forward chemical genetics screen designed to identify small molecule inhibitors of endothelial morphogenesis. This screen resulted in the identification of structurally-related small molecules that inhibited endothelial cell proliferation, migration, the ability to form tubule-like structures in matrigel, as well as neo- angiogenesis visualized using a dorsal skin fold vascular window chamber. Biochemical analysis revealed that the novel small molecules inhibited ENOX1, a plasma membrane- associated enzyme that exhibits protein disulfide-thiol interchange activity. While ENOX activity is important for cellular proliferation, the molecular mechanisms are not well understood. shRNA-mediated inhibition of ENOX activity in HUVECs inhibited endothelial cell migration and formation of tubule-like structures in matrigel, reproducing the effects of the small molecule inhibitors. Small molecule inhibition of ENOX1 was associated with a significant increase in endothelial cell apoptosis induced by ionizing radiation. Colony formation assays demonstrated that these small molecules increased the radiation sensitivity of endothelial cells. In contrast, the radiation sensitivity of tumor epithelial cells was unaffected by the small molecules. Administration of a small molecule ENOX1 inhibitor prior to fractionated X-irradiation produced a statistically significant decrease in the number and density of CD34 expressing Lewis Lung Carcinoma (LLC) tumor-associated microvasculature. Use of LLC and human HT29 colon carcinoma tumor models demonstrated that the small molecule ENOX1 inhibitors coupled with fractionated X-irradiation produced a statistically significant increase in tumor growth delay compared to irradiation alone. No evidence of acute toxicity was observed over a 30 day interval when mice received a bolus injection of 120 mg/kg. These preliminary data support the hypothesis that ENOX1 represents a rationale molecular target for increasing the ability of ionizing radiation to control tumor growth. The short term goal of this application is to valid this hypothesis. The long term goal is to develop these small molecule inhibitors into a radiation sensitizer that exhibits clinical efficacy. PUBLIC HEALTH RELEVANCE: A forward chemical genetics screen identifies small molecule inhibitors of endothelial morphogenesis. Biochemical analysis revealed that these novel small molecules inhibited the enzyme ENOX1, increased the radiation sensitivity of endothelial cells, increased radiation- mediated suppression of tumor-associated vasculature, thereby suppressing tumor growth. These preliminary data support the hypothesis that ENOX1 represents a rationale molecular target for increasing the ability of ionizing radiation to control tumor growth.