Abstract
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.
Footnotes
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This work was supported by National Institutes of Health grants GM066119 (to P.S.T.), MH081265 (to P.S.T.), and MH074404 (to H.R.).
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This is publication 18911-IMM from the Department of Immunology, The Scripps Research Institute.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.107.038349.
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ABBREVIATIONS: TLR, Toll-like receptor; HTS, high-throughput screening; DMSO, dimethyl sulfoxide; LPS, lipopolysaccharide; TIR domain, Toll/interleukin-1 receptor homology domain; ELISA, enzyme-linked immunosorbent assay; HEK, human embryonic kidney; DMEM, Dulbecco's modified Eagle's medium; IL, interleukin; pIpC, polyinosinic-polycytidylic acid; TNF-α, tumor necrosis factor α; NF-κB, nuclear factor κB; FACS, fluorescence-activated cell sorting; PBS, phosphate-buffered saline; HA, hemagglutinin; FCS, fetal calf serum; AM, acetoxymethyl ester; TIRAP, toll-interleukin 1 receptor domain containing adaptor protein; S/B, signal-to-background ratio; MALP-2, macrophage-activating lipopeptide 2 kDa; CpG, cytosine phosphate guanine; TLR4CD, Toll-like receptor 4 transmembrane-cytoplasmic domain; Bla, β-lactamase.
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The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material. - Received May 21, 2007.
- Accepted July 5, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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Application of β-Lactamase Enzyme Complementation to the High-Throughput Screening of Toll-Like Receptor Signaling Inhibitors
