RT Journal Article
SR Electronic
T1 Application of β-Lactamase Enzyme Complementation to the High-Throughput Screening of Toll-Like Receptor Signaling Inhibitors
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 868
OP 875
DO 10.1124/mol.107.038349
VO 72
IS 4
A1 Hyun-Ku Lee
A1 Steven J. Brown
A1 Hugh Rosen
A1 Peter S. Tobias
YR 2007
UL http://molpharm.aspetjournals.org/content/72/4/868.abstract
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