A homogeneous time-resolved fluorescence-based high-throughput screening system for discovery of inhibitors of...

The nuclear transcription factor NF-kappaB is crucial to the expression of numerous cytokines, enzymes, and cell adhesion molecules, all of which can drive inflammatory and autoimmune disorders such as rheumatoid arthritis. The IKK complex plays the most important role in the signal cascade leading to NF-kappaB activation. Recently, inhibition of the interaction between NEMO (NF-kappaB essential modulator) and the catalytic subunits of IKK, especially IKKbeta, has received particular attention as a possible new therapeutic approach to treatment of inflammatory disorders, and several reports have shown the efficacy of cell permeable NEMO binding domain (NBD)-containing peptides in blocking the IKK/NF-kappaB pathway. In this article, we describe in detail the development and validation of two novel binding assays, a homogeneous time-resolved fluorescence (HTRF)-based assay and an enzyme-linked immunosorbent assay (ELISA)-based assay, suitable for the discovery of small molecules that inhibit IKKbeta-NEMO interaction. Using the HTRF-based assay, we screened approximately 15,000 compounds from our chemical library and eliminated false positive hits by the ELISA-based assay and IKK complex kinase assay. As a result, seven positive hit compounds that inhibit IKK complex activity through inhibition of IKKbeta-NEMO interaction were identified. These hit compounds may have a good potential in the treatment of inflammatory and autoimmune disorders such as rheumatoid arthritis.