Generation and utility of NADPH oxidase inhibitors

描述（由申请人提供）：本研究是根据NIH通知号PA-07-320（开发高通量药物筛选测定法）提出的。我们已经发现抗氧化剂能明显抑制癌细胞的侵殖形成。与未转化的癌细胞相比，在癌细胞中检测到更高水平的ROS，并且令人惊讶的是，其中一些ROS定位于侵过足。由NADPH氧化酶系统产生的ROS是侵殖体形成和功能所必需的。在结构上与p47phox（吞噬细胞中的NADPH氧化酶成分）相关的侵足蛋白Tks5的敲低可降低癌细胞中的总ROS水平。Tks5促进了侵殖体形成所需的ROS的产生，反过来，ROS在一个正反馈回路中调节Tks5酪氨酸磷酸化。人类基因组包含几个NADPH氧化酶催化亚基，称为Nox1， Nox2, Nox3， Nox4和Nox5。在评估Nox亚基在人类癌细胞中的表达时，我们发现Nox4和Nox1是最常表达的。此外，敲低Nox4或Nox1可抑制侵殖细胞的形成和侵袭行为。这些数据使我们假设NADPH氧化酶代表了治疗播散性癌症的新治疗靶点。测试这一假设的最佳方法是使用小分子抑制剂，但没有一种可用的抑制剂具有适当的选择性和药物特性。我们建议产生Nox选择性抑制剂，以便在体内研究Nox在癌症进展中的作用。我们将：建立和验证基于细胞的Nox活性分析，以及特异性分析；进行高通量筛选并验证命中；并提高效力和选择性，以产生工具化合物，在体外和体内的假设检验。这项研究的意义在于有可能确定治疗发展的新途径。这项研究的直接影响是，它代表了新的治疗靶点在体内验证的重要和必要的第一步。

DESCRIPTION (provided by applicant): This research is proposed in response to NIH Notice Number PA-07-320 (Development of assays for high-throughput drug screening). We have found that anti-oxidants acutely inhibit invadopodia formation in cancer cells. Higher levels of ROS are detected in cancer cells, compared to their non-transformed counterparts, and strikingly some of this ROS is localized in invadopodia. ROS generated by the NADPH oxidase system are necessary for invadopodia formation and function. Knockdown of the invadopodia protein Tks5, which is structurally related to p47phox (an NADPH oxidase component in phagocytic cells) reduces total ROS levels in cancer cells. Tks5 facilitates the production of ROS necessary for invadopodia formation, and in turn ROS modulates Tks5 tyrosine phosphorylation in a positive feedback loop. The human genome contains several NADPH oxidase catalytic subunits, called Nox1, Nox2, Nox3, Nox4 and Nox5. In evaluating the expression of the Nox subunits in human cancer cells, we found that Nox4 and Nox1 are most frequently expressed. Furthermore, knockdown of Nox4 or Nox1 inhibits invadopodia formation and invasive behavior. These data lead us to hypothesize that NADPH oxidases represent novel therapeutic targets for the treatment of disseminated cancers. Testing this hypothesis will be best accomplished using small molecule inhibitors, yet none of available inhibitors have the appropriate selectivity and pharmaceutical properties. We propose to generate Nox selective inhibitors to allow in vivo studies on the role of Nox in cancer progression. We will: establish and validate cell-based assays for Nox activity, as well as specificity assays; conduct a high throughput screen and verify hits; and improve potency and selectivity, in order to generate tool compounds for hypothesis testing in vitro and in vivo. The significance of this research lies in the possibility of identifying a new avenue for therapeutic development. The immediate impact of this research is that it represents an important and necessary first step in the in vivo validation of a new therapeutic target. PUBLIC HEALTH RELEVANCE: The growth of disseminated cancers leads to morbidity and mortality, and there is an urgent unmet need for new therapeutic targets. This research seeks to develop antagonists of an enzyme that mediates invasive behavior of cancer cells, and thus is of relevance to the public health problem of metastatic cancer.