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.
Relevance to Public Health: The inflammasome is emerging as an important mediator of numerous diseases. The studies proposed in this project have potential for developing novel drugs that can be used as therapies for many inflammatory diseases, including gout, arthritis, Alzheimer's disease, type 2 diabetes mellitus, atherosclerosis, silicosis, and pulmonary fibrosis. Successful completion of this project will lead to the development of new inflammasome inhibitors that will be subsequently tested in several preclinical models of disease.
