HTS for FADD kinase inhibitors using molecular imaging

描述(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，我们最近发现了一个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、头颈部、脑和成年男性生殖细胞肿瘤。对肺癌的研究表明，FADD的过度表达与不良的临床结果显著相关。基于免疫组织化学的组织芯片分析证实了FADD过度表达与预后不良的关系，同时也揭示了核定位磷酸化FADD(p-FADD)的存在。P-FADD表达增加的肿瘤也表现为核因子？B活化增强。综上所述，我们实验室和其他实验室发表的结果表明，FADD的磷酸化和核因子-β激活之间存在因果关系，这是侵袭性治疗耐药癌症表型的特征。因此，我们推测抑制肿瘤细胞中FADD的磷酸化可能会使肿瘤细胞对化疗药物敏感。为了帮助这一假说的实验，我们求助于分子成像工具，并开发了一种PAN FADD激酶报告(FKR)，它可以非侵入性地实时检测FADD-激酶的活性。在具体目标1中，我们将表征FKR的敏感性和特异性。在特定的目标2A中，我们将进行高通量筛选，从一组不同的化合物文库中识别以FADD磷酸化为靶点的分子。利用表达突变FKR或荧光素酶的细胞的二级筛选，有毒和不那么敏感的铅分子将被消除。在特定目标2B中，我们将通过量化顶端引线的IC50来评估候选分子的相对有效性。在特定的目标2C中，将使用蛋白质印迹和蛋白激酶阵列来研究候选分子抑制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接下来的几年里进行研究。

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.