title: Application of beta-lactamase enzyme complementation to the high-throughput screening of toll-like receptor signaling inhibitors.
abstract: We describe a successful application of beta-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 beta-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 beta-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 microM.
authors.affiliation: Department of Immunology, IMM-12, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. tobias@scripps.edu
authors.forename: Hyun-Ku
authors.initials: HK
authors.lastname: Lee
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Brown
authors.forename: Hugh
authors.initials: H
authors.lastname: Rosen
authors.forename: Peter S
authors.initials: PS
authors.lastname: Tobias
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D024202:Electrophoretic Mobility Shift Assay; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D053594:Myeloid Differentiation Factor 88 / Q000037:antagonists & inhibitors; D011485:Protein Binding; D015398:Signal Transduction / Q000187:drug effects*; D051197:Toll-Like Receptor 4 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001618:beta-Lactamases / Q000378:metabolism*
chemical_list: C507780:Myd88 protein, mouse; D053594:Myeloid Differentiation Factor 88; D051197:Toll-Like Receptor 4; D001618:beta-Lactamases