HTS for FADD kinase inhibitors using molecular imaging

描述（由申请人提供）：利用差异表达谱、定量二维（2d）凝胶电泳和数据挖掘，我们最近发现了一种新的预后生物标志物，fas相关死亡结构域（FADD），它在许多人类恶性肿瘤中过表达，如肺、头颈、脑和成年男性生殖细胞肿瘤。肺癌研究表明，FADD过表达与不良临床预后显著相关。基于免疫组织化学的组织微阵列分析证实了FADD过表达与不良预后之间的关联，并且还发现了核定位磷酸化FADD （p-FADD）的存在。p-FADD表达增加的肿瘤也显示NF-？B激活。综上所述，我们实验室和其他实验室发表的结果表明，FADD的磷酸化和NF-？B活化，一个积极的治疗抵抗癌症表型的标志。因此，我们假设抑制肿瘤细胞中的FADD磷酸化可能会使癌细胞对化疗药物敏感。为了帮助这一假设的实验，我们利用分子成像工具，开发了一种pan FADD激酶报告器（FKR），它可以无创地实时感知FADD激酶的活性。在Specific Aim 1中，我们将描述FKR的敏感性和特异性。在Specific Aim 2A中，我们将执行高通量筛选，以识别针对FADD磷酸化的各种化合物文库中的分子。利用表达突变体FKR或荧光素酶的细胞进行二次筛选，将消除有毒且不太敏感的铅分子。在Specific Aim 2B中，我们将通过量化顶端导联的IC50来评估候选分子的相对功效。在Specific Aim 2C中，候选分子抑制FADD激酶的特异性将使用western blotting和蛋白激酶阵列进行研究。这些化合物及其衍生物在癌症治疗中的效用将在随后几年进行研究。

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.