Development of inlammasome inhibitors to be used as anti-inflammatory agents

描述（由申请人提供）：炎症是许多疾病的中心病因组成部分，抗炎药物，如皮质类固醇，是对抗许多疾病的重要疗法。然而，许多疾病对目前的抗炎治疗没有充分的反应。因此需要新的抗炎疗法。炎性小体是最近发现的先天免疫反应的一个分支，它越来越被认为是许多炎症性疾病的重要决定因素。炎性小体是一种多蛋白复合物，可对多种刺激（包括细菌、病毒、二氧化硅、石棉、胆固醇和尿酸）作出反应，启动由细胞因子IL-1b、IL-18和IL-33介导的促炎反应。炎性小体的不当激活与多种疾病有关，包括痛风、关节炎、糖尿病、阿尔茨海默病、肺纤维化和动脉粥样硬化。炎性小体在其他疾病中的作用仍在不断发展，该途径的抑制剂将为许多疾病提供潜在的治疗方法，并将提供有助于解剖该途径的机制研究。到目前为止，已经确定了四种炎性小体复合物，每种炎性小体对分子激活剂的独特组合有反应。然而，所有炎性小体的一个核心组成部分是其激活和分泌细胞因子IL-1b、IL-18和IL-33。因此，IL-1b的分泌是炎症小体激活的合适标记物，而发现新的炎症小体抑制剂将为大量炎症性疾病提供潜在的治疗方法。二级和三级研究将更直接地测量炎性体复合体蛋白caspase-1的活性。具体目的：利用高通量的细胞为基础的分析，以确定炎性小体的小分子抑制剂。本提案将重点筛选化合物以鉴定IL-1b抑制剂，作为炎症小体活性的标志物。我们开发了一种高通量1536孔的检测方法，使用AlphaLISA IL-1b试剂盒（Perkin Elmer）来定量人单核细胞系THP-1的IL-1b分泌。我们的初步数据表明，我们可以很容易地检测到活化THP-1细胞的IL-1b分泌，并可以量化已知炎症小体抑制剂的IC50值。我们已经与NIH化学基因组学中心（NCGC）建立了合作关系，我们的化学文库将来自他们的分子文库计划。这个文库由一组扩展的小分子组成，总共超过30万种化合物。通过初级筛选确定的药物将通过二级和三级筛选进行验证和表征，以测量caspase-1和NF-kB的活性，以及其他炎性小体依赖和独立细胞因子的分泌。Hit化合物将在缺乏各种炎症小体成分的小鼠细胞中进行验证，后续研究将检验体内疗效。该研究的成功完成将产生抑制细胞系统中炎性体的新化合物。

DESCRIPTION (provided by applicant): Inflammation is a central etiological component of numerous diseases, and anti-inflammatory drugs, such as corticosteroids, represent important therapies for combating a large number of diseases. However, many diseases do not adequately respond to current anti-inflammatory therapies. Thus new anti-inflammatory therapies are needed. The inflammasome is a recently identified arm of the innate immune response, which is becoming increasingly recognized as an important determinant of numerous inflammatory diseases. The inflammasome is a mult-protein complex that responds to numerous stimuli, including bacteria, viruses, silica, asbestos, cholesterol, and uric acid, to initiate a pro-inflammatory response that is mediated by the cytokines IL-1b, IL-18, and IL-33. Improper activation of the inflammasome has been implicated in multiple diseases, including gout, arthritis, diabetes, Alzheimer's, pulmonary fibrosis, and atherosclerosis. The role of the inflammasome in other disorders is still evolving, and inhibitors of this pathway will provide potential therapeutics for numerous diseases, and will provide mechanistic studies that will aid in dissection of this pathway. There are four inflammasome complexes identified thus far, and each inflammasome responds to a unique combination of molecular activators. However, a central component of all inflammasomes is its activation and secretion of the cytokines IL-1b, IL-18, and IL-33. Thus, secretion of IL-1b is a suitable marker for inflammasome activation, and identifying novel inhibitors of the inflammasome will provide potential therapies for a large number of inflammatory diseases. Secondary and tertiary studies will more directly measure activity of the inflammasome complex protein caspase-1. Specific Aim: To utilize a high throughput cell-based assay to identify small molecule inhibitors of the inflammasome. This proposal will focus on screening compounds to identify inhibitors of IL-1b, as a marker of inflammasome activity. We have developed a high throughput 1536-well assay using the AlphaLISA IL1b Kit (Perkin Elmer) to quantify IL-1b secretion from the human monocyte cell line THP-1. Our preliminary data demonstrates that we can readily detect IL-1b secretion from activated THP-1 cells and can quantify IC50 values of known inflammasome inhibitors. We have established a collaboration with the NIH Chemical Genomics Center (NCGC), and our chemical library will be derived from their Molecular Libraries Program. This library consists of an expansive set of small molecules totaling greater than 300,000 compounds. Drugs identified by this primary screen will be validated and characterized by secondary and tertiary screens to measure activity of caspase-1 and NF-kB, and secretion of other inflammasome-dependent and independent cytokines. Hit compounds will be validated in cells from mice deficient in various components of the inflammasome, and follow-up studies will examine efficacy in vivo. Successful completion of the proposed study will yield novel compounds that inhibit the inflammasome in cell-based systems.