The specific aim of this proposal is to use a high throughput screening assay that we have developed to find inhibitors of TLR4 signaling. There are a number of diseases whose etiology involves chronic or acute, but sterile, inflammation. Examples are atherosclerosis and kidney transplantation. In some of these diseases, notably the two just mentioned, inflammation initiated by Toll-like receptor (TLR) signaling is clearly involved. However, there are no inhibitors of TLR signaling that might serve as models for small molecule inhibitors of the activity of TLRs and which might be useful, or serve as starting points for development of therapeutic compounds. We have developed an assay that exhibits a positive signal when TLR4 and its intracellular signaling adapter MyD88 associate. The signal develops when two fragments of ?-lactamase, expressed as chimeric proteins with TLR4 and MyD88, reconstitute ?-lactamase activity when the two chimeric proteins associate. Thus the assay measures ?-lactamase activity indicating TLR4 - MyD88 association. Inhibition of the ?-lactamase activity signals detection of an inhibitor of TLR4 - MyD88 association. Following high throughput screening with this assay we will eliminate false positive hits by assaying for ?-lactamase inhibitors. We will also assay positive hits for their ability to inhibit TLR4-MyD88 association by co-immunoprecipitation assays. Finally we will assess for effects of the positive hit on association of MyD88 with other TLRs. There are a number of human diseases, such as atherosclerosis and kidney disease, which involve inflammation generated by the activity of cell surface proteins called Toll-like receptors (TLRs). There are no good candidates for drugs that would block the activity of TLRs that could be useful in treating these diseases. This project will utilize an assay that we have recently developed to search for such compounds. ? ? ?
| Lee, Hyun-Ku; Brown, Steven J; Rosen, Hugh et al. (2007) Application of beta-lactamase enzyme complementation to the high-throughput screening of toll-like receptor signaling inhibitors. Mol Pharmacol 72:868-75 |
