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Molecular Pharmacology

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Research ArticleArticle

Application of β-Lactamase Enzyme Complementation to the High-Throughput Screening of Toll-Like Receptor Signaling Inhibitors

Hyun-Ku Lee, Steven J. Brown, Hugh Rosen and Peter S. Tobias
Molecular Pharmacology October 2007, 72 (4) 868-875; DOI: https://doi.org/10.1124/mol.107.038349
Hyun-Ku Lee
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Steven J. Brown
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Hugh Rosen
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Peter S. Tobias
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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

  • 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.).

  • This is publication 18911-IMM from the Department of Immunology, The Scripps Research Institute.

  • Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

  • doi:10.1124/mol.107.038349.

  • 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.

  • ↵ Embedded Image 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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Molecular Pharmacology: 72 (4)
Molecular Pharmacology
Vol. 72, Issue 4
1 Oct 2007
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Research ArticleArticle

Application of β-Lactamase Enzyme Complementation to the High-Throughput Screening of Toll-Like Receptor Signaling Inhibitors

Hyun-Ku Lee, Steven J. Brown, Hugh Rosen and Peter S. Tobias
Molecular Pharmacology October 1, 2007, 72 (4) 868-875; DOI: https://doi.org/10.1124/mol.107.038349

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Research ArticleArticle

Application of β-Lactamase Enzyme Complementation to the High-Throughput Screening of Toll-Like Receptor Signaling Inhibitors

Hyun-Ku Lee, Steven J. Brown, Hugh Rosen and Peter S. Tobias
Molecular Pharmacology October 1, 2007, 72 (4) 868-875; DOI: https://doi.org/10.1124/mol.107.038349
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