HTS for FADD kinase inhibitors using molecular imaging

描述（由申请人提供）：使用差异表达谱、定量二维（2-D）凝胶电泳和数据挖掘，我们最近鉴定了一种新的预后生物标志物，Fas相关死亡结构域（FADD），其在许多人类恶性肿瘤中过表达，如肺、头颈部、脑和成人男性生殖细胞肿瘤。肺癌研究显示，FADD的过度表达与不良临床结局显著相关。基于免疫组织化学的组织微阵列分析证实了FADD过表达和不良结局之间的关联，并且还揭示了核定位磷酸化FADD（p-FADD）的存在。肿瘤与增加p-FADD表达也表现出升高NF-？B激活。两者合计，从我们的实验室和其他人发表的结果表明FADD和NF-？B活化，侵袭性治疗抗性癌症表型的标志。因此，我们假设抑制肿瘤细胞中的FADD磷酸化可以使癌细胞对化疗剂敏感。为了帮助这个假设的实验，我们已经求助于分子成像工具，并开发了一个泛FADD激酶报告（FKR），非侵入性传感FADD-激酶活性在真实的时间。在具体目标1中，我们将描述FKR的灵敏度和特异性。在Specific Aim 2A中，我们将进行高通量筛选，以从靶向FADD磷酸化的不同化合物文库中鉴定分子。利用表达突变FKR或荧光素酶的细胞进行二次筛选，将消除毒性和较不敏感的先导分子。在特定目标2B中，我们将通过定量最佳先导化合物的IC 50来评价候选分子的相对疗效。在特异性目标2C中，将使用蛋白质印迹和蛋白激酶阵列研究候选分子抑制FADD激酶的特异性。这些化合物及其衍生物在癌症治疗中的效用将在随后的几年中进行研究。

DESCRIPTION (provided by applicant): Using differential expression profiling, quantitative two-dimensional (2-D) gel electrophoresis and data mining we recently identified a new prognostic biomarker, Fas-associated death domain (FADD), which is overexpressed in a number of human malignancies such as lung, head and neck, brain and adult male germ cell tumors. Studies in lung cancer revealed that overexpression of FADD significantly associated with poor clinical outcome. Immunohistochemistry-based tissue microarray analysis confirmed the association between FADD over-expression and the poor outcome, and also revealed the presence of nuclear localized phosphorylated FADD (p-FADD). Tumors with increased p-FADD expression also showed elevated NF-?B activation. Taken together, published results from our lab and others suggest a causal relationship between the phosphorylation of FADD and NF-?B activation, a hallmark of an aggressive therapy resistant cancer phenotype. Thereby, we hypothesize that inhibiting FADD phosphorylation in tumor cells may sensitize cancer cells to chemotherapeutic agents. To aid in experimentation of this hypothesis we have resorted to molecular imaging tools and developed a pan FADD kinase reporter (FKR) which non-invasively senses FADD-kinase activity in real time. In Specific Aim 1, we will characterize the sensitivity and specificity of FKR. In Specific Aim 2A we will perform a high throughput screen to identify molecules from a diverse set of compound libraries that target FADD phosphorylation. Utilizing secondary screens with cells expressing either mutant FKR or luciferase, the toxic and less sensitive lead molecules will be eliminated. In Specific Aim 2B we will evaluate the relative efficacy of the candidate molecules by quantifying IC50 of the top leads. In Specific Aim 2C the specificity of candidate molecules in inhibiting FADD kinases will be investigated using western blotting and protein kinase arrays. The utility of these compounds and their derivatives in the treatment of cancers will be investigated in subsequent years.