Novel targets in the regulation of NF-kB and SREBP activity: A two-faceted approa

描述（由申请人提供）：近年来，糖尿病已成为一种日益严重的流行病，CDC估计仅在美国就有2400万人直接受影响。有两个相互关联的途径在糖尿病的发病中至关重要。它们是营养来源的代谢途径和促炎途径。通常发现上述两种途径的异常激活与糖尿病的病理进展相关。我们已经确定了两个新的转录激活因子-介体相互作用，控制和调节代谢和炎症途径。两种转录激活因子NF-：B（核因子-：B）和SREBP（固醇调节元件结合蛋白）在糖尿病的发生和发展中起重要作用。这两种因子已被证明在功能上与被称为激活剂募集辅因子（ARC）/介体复合物的辅激活剂相互作用。NF-：B和SREBP在调节参与糖尿病发病的许多基因的表达中是关键的。我们建议用小分子效应物靶向这两种转录因子（SREBP和NF-：B）的激活剂-介体相互作用，从而抑制基因激活。SREBP和NF-：B的激活剂-介体相互作用代表了新的靶点，因为它们的抑制会影响其激活途径中的最下游元件。我们将通过溶液状态NMR确定构成相互作用的最小元素的结构（即，介体中的KIX结构域与激活剂的反式激活结构域（KIX）的复合物）。我们将通过高通量筛选确定每个激活剂-介体相互作用的小分子抑制剂，并在体外转录测定中验证它们。随后，我们将研究这些抑制剂对基因转录和脂肪细胞分化的影响，在体内（细胞培养），在3 T3-L1前脂肪细胞。这将形成未来研究的基础，其中本文鉴定和表征的抑制剂将在秀丽隐杆线虫和糖尿病小鼠模型中进行测试。此外，我们试图阐明一种新的机制，SREBP靶基因的调控精心策划的let-7 micro-RNA。已知微RNA调节一组基因的mRNA水平，从而协同调节整个途径。我们假设let-7调节PGC-1共激活因子的水平，PGC-1共激活因子反过来调节SREBP靶基因，并且预计在脂肪生成和线粒体生物生成中具有深远的意义。 公共卫生相关性：近年来，糖尿病已成为一种日益严重的流行病。营养来源的代谢途径和促炎途径在糖尿病的发病中至关重要，我们已经确定了两种新的转录激活因子-介体相互作用，控制和调节这些途径。我们打算通过识别激活剂-介体相互作用的小分子抑制剂来靶向这些途径中的基因激活，并了解这些途径中微小RNA的内源性基因调节。

DESCRIPTION (provided by applicant): Diabetes has become an increasingly serious epidemic in recent years, with CDC estimates of 24 million people directly affected in the United States alone. There are two interconnected pathways that are critical in the onset of diabetes. They are the nutritionally-derived metabolic pathway, and the pro-inflammatory pathway. Aberrant activation of both the above pathways is commonly found to be associated with the pathological progression of diabetes. We have identified two novel transcription activator-Mediator interactions that control and regulate the metabolic and inflammatory pathway. The two transcription activators, NF-:B (Nuclear Factor- :B) and SREBP (Sterol Regulatory Element-Binding Protein), play significant roles in the onset and progression of diabetes. Both these factors have been shown to functionally interact with a co-activator known as the Activator Recruited Co-factor(ARC)/Mediator Complex. NF-:B and SREBP are critical in regulating the expression of a number of genes involved in the onset of diabetes. We propose to target the activator-Mediator interaction of these two transcription factors (SREBP and NF-:B) with small molecular effectors, thus inhibiting gene activation. The activator-Mediator interactions of SREBP and NF-:B represent novel targets because their inhibition would affect the most downstream element in their activation pathways. We will determine the structure of the minimal elements that constitute the interaction (i.e. the complex of KIX domain in the Mediator with the transactivation domain (TAD) of the activator) by solution state NMR. We will identify small molecule inhibitors for each of the activator-Mediator interactions by high-throughput screening and validate them in an in vitro transcription assay. We will subsequently study the effect of these inhibitors on gene transcription and adipocyte differentiation, in vivo (cell culture), in 3T3-L1 pre-adipocyte cells. This will form the basis for future studies wherein the inhibitors identified and characterized here, will be tested in C.elegans and diabetic murine models. In addition, we seek to elucidate a novel mechanism of regulation of SREBP target genes orchestrated by let-7 micro-RNA. Micro-RNAs are known to modulate mRNA levels of a set of genes, thus synergistically regulating an entire pathway. We hypothesize let-7 modulates levels of PGC-1 co-activators, which in turn regulates SREBP target genes, and is expected to have profound implications in lipogenesis and mitochondrial biogenesis. PUBLIC HEALTH RELEVANCE: Diabetes has become an increasingly serious epidemic in recent years. The nutritionally-derived metabolic pathway and the pro-inflammatory pathways are critical in the onset of diabetes and we have identified two novel transcription activator-Mediator interactions that control and regulate these pathway. We intend to target gene activation in these pathways by identifying small molecule inhibitors for the activator-Mediator interactions and understand endogenous gene regulation by micro-RNAs in these pathways.