PT - JOURNAL ARTICLE AU - Hyun-Ku Lee AU - Steven J. Brown AU - Hugh Rosen AU - Peter S. Tobias TI - Application of β-Lactamase Enzyme Complementation to the High-Throughput Screening of Toll-Like Receptor Signaling Inhibitors AID - 10.1124/mol.107.038349 DP - 2007 Oct 01 TA - Molecular Pharmacology PG - 868--875 VI - 72 IP - 4 4099 - http://molpharm.aspetjournals.org/content/72/4/868.short 4100 - http://molpharm.aspetjournals.org/content/72/4/868.full SO - Mol Pharmacol2007 Oct 01; 72 AB - We describe a successful application of β-lactamase fragment complementation to high-throughput screening (HTS) for Toll-like receptor 4 (TLR4) signaling inhibitors. We developed a stable cell line, HeLa/CL3-4, expressing MyD88/Bla(a) and TLR4/Bla(b), in which the two β-lactamase fragments complement with each other by virtue of spontaneous MyD88-TLR4 binding via their Toll/IL-1R (TIR) domains. Inhibition of the MyD88-TLR4 binding leads to the disruption of the enzyme complementation and a loss of the lactamase activity. We used a 384-well plate format to screen 16,000 compounds using this assay and obtained 45 primary hits. After rescreening these 45 hits and eliminating compounds that directly inhibited β-lactamase, we had five candidate inhibitors. We show that these five act as inhibitors of TLR4-MyD88 binding and are variously effective at inhibiting lipopolysaccharide-stimulated cytokine release from RAW264.7 cells. One compound is effective near 100 nM. None of the compounds showed any cytotoxicity at 20 μM. The American Society for Pharmacology and Experimental Therapeutics