Identification of novel scaffolds for IkappaB kinase beta inhibitor via a high-throughput screening TR-FRET assay.

摘要

Control of NF-kappaB release through the inhibition of IkappaB kinase beta (IKKbeta) has been identified as a potential target for the treatment of inflammatory and autoimmune diseases. To screen the small molecule compound library against IKKbeta, a high-throughput screening (HTS) campaign was carried out using immobilized metal affinity for phosphochemicals (IMAP)-based time-resolved fluorescence resonance energy transfer (TR-FRET) assay. Through serial optimization of assay conditions, the Z' value was achieved at 0.88 from the pilot library screening of the most diverse 7,243 compounds with reconfirmation rate of 63%. The results from this HTS campaign identified three novel scaffolds for the prospective IKKbeta inhibitor, such as 7-benzoyl-4-phenylcyclopenta[1,2] oxazine, 1-(thiophen or furan)-2,3-dihydroimidazo[1,5] pyridine and 2-phenyloxazolo[5,4] pyridine. Particularly, 7-benzoyl-4-phenylcyclopenta[1,2] oxazine derivatives presented potent inhibitory activity and selectivity for IKKbeta. These findings suggest that the current TR-FRET assay system for IKKbeta was successful to identify hits for novel IKKbeta inhibitors as a robust, reproducible and sensitive HTS system.