title: High-throughput screening for TLR3-IFN regulatory factor 3 signaling pathway modulators identifies several antipsychotic drugs as TLR inhibitors.
abstract: TLR3 is one of the major innate immune sensors of dsRNA. The signal transduction pathway activated by TLR3, upon binding to dsRNA, leads to the activation of two major transcription factors: NF-kappaB and IFN regulatory factor (IRF) 3. In an effort to identify specific chemical modulators of TLR3-IRF3 signal transduction pathway, we developed a cell-based readout system. Using the IFN-stimulated gene 56 promoter-driven firefly luciferase gene stably integrated in a TLR3-expressing HEK293 cell line, we were able to generate a cell line where treatment with dsRNA resulted in a dose-dependent induction of luciferase activity. A screen of two pharmacologically active compound libraries using this system identified a number of TLR3-IRF3 signaling pathway modulators. Among them we focused on a subset of inhibitors and characterized their mode of action. Several antipsychotic drugs, such as sertraline, trifluoperazine, and fluphenazine, were found to be direct inhibitors of the innate immune signaling pathway. These inhibitors also showed the ability to inhibit IFN-stimulated gene 56 induction mediated by TLR4 and TLR7/8 pathways. Interestingly, they did not show significant effects on TLR3-, TLR7-, and TLR8-mediated NF-kappaB activation. Detailed analysis of the signaling pathway indicated that these drugs might be exerting their inhibitory effects on IRF3 via PI3K signaling pathway. The data presented in this study provide mechanistic explanation of possible anti-inflammatory roles of some antipsychotic drugs.
authors.affiliation: Molecular Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213, USA.
authors.forename: Jianzhong
authors.initials: J
authors.lastname: Zhu
authors.forename: Kevin
authors.initials: K
authors.lastname: Smith
authors.forename: Paishiun N
authors.initials: PN
authors.lastname: Hsieh
authors.forename: Yvonne K
authors.initials: YK
authors.lastname: Mburu
authors.forename: Saurabh
authors.initials: S
authors.lastname: Chattopadhyay
authors.forename: Ganes C
authors.initials: GC
authors.lastname: Sen
authors.forename: Saumendra N
authors.initials: SN
authors.lastname: Sarkar
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D014150:Antipsychotic Agents / Q000494:pharmacology*; D002460:Cell Line; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D002999:Clone Cells; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D050838:Interferon Regulatory Factor-3 / Q000037:antagonists & inhibitors* / Q000502:physiology*; D009025:Morpholines / Q000494:pharmacology; D012330:RNA, Double-Stranded / Q000037:antagonists & inhibitors / Q000494:pharmacology; D016601:RNA-Binding Proteins; D020280:Sertraline / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects* / Q000276:immunology*; D051196:Toll-Like Receptor 3 / Q000037:antagonists & inhibitors* / Q000502:physiology*; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D014268:Trifluoperazine / Q000494:pharmacology
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D014150:Antipsychotic Agents; D002867:Chromones; C484290:IFIT1 protein, human; D050838:Interferon Regulatory Factor-3; D009025:Morpholines; D012330:RNA, Double-Stranded; D016601:RNA-Binding Proteins; C495344:TLR3 protein, human; D051196:Toll-Like Receptor 3; D014157:Transcription Factors; D014268:Trifluoperazine; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D020280:Sertraline
references.citation: J Biol Chem. 2002 Aug 30;277(35):32124-32
references.pmid: 12052830
references.citation: Genes Dev. 2005 Nov 15;19(22):2668-81
references.pmid: 16260493
references.citation: J Biol Chem. 2007 Dec 21;282(51):36953-60
references.pmid: 17932028
references.citation: Nat Struct Mol Biol. 2009 Jun;16(6):652-7
references.pmid: 19430461
references.citation: Mol Immunol. 2008 May;45(10):2790-6
references.pmid: 18339426
references.citation: Nature. 2001 Oct 18;413(6857):732-8
references.pmid: 11607032
references.citation: Science. 2007 Sep 7;317(5843):1390-3
references.pmid: 17690258
references.citation: Nat Med. 2005 Apr;11(4 Suppl):S63-8
references.pmid: 15812492
references.citation: Science. 2005 Sep 16;309(5742):1857-61
references.pmid: 16166517
references.citation: Cell. 2006 Feb 24;124(4):783-801
references.pmid: 16497588
references.citation: J Exp Med. 2008 Feb 18;205(2):315-22
references.pmid: 18227218
references.citation: J Immunol. 2008 Feb 15;180(4):2054-61
references.pmid: 18250410
references.citation: Immunol Res. 2006;34(1):33-48
references.pmid: 16720897
references.citation: Pharmacol Ther. 2004 Sep;103(3):245-59
references.pmid: 15464592
references.citation: Proc Natl Acad Sci U S A. 2002 Apr 30;99(9):6322-7
references.pmid: 11972054
references.citation: Nat Immunol. 2003 May;4(5):491-6
references.pmid: 12692549
references.citation: Curr Opin Immunol. 2008 Feb;20(1):17-22
references.pmid: 18272355
references.citation: J Biomol Screen. 1999;4(2):67-73
references.pmid: 10838414
references.citation: Nat Med. 2004 Dec;10(12):1366-73
references.pmid: 15558055
references.citation: J Mol Neurosci. 2006;29(3):185-94
references.pmid: 17085778
references.citation: J Leukoc Biol. 2009 Jun;85(6):966-77
references.pmid: 19289601
references.citation: J Biol Chem. 2001 Aug 10;276(32):30178-82
references.pmid: 11487589
references.citation: J Immunol. 2008 Jan 1;180(1):483-91
references.pmid: 18097050
references.citation: Respir Res. 2007 May 03;8:35
references.pmid: 17477857
references.citation: Nature. 2006 Jan 12;439(7073):208-11
references.pmid: 16306936
references.citation: Cell. 2006 Feb 24;124(4):823-35
references.pmid: 16497591
references.citation: Immunology. 2006 Jan;117(1):11-21
references.pmid: 16423036
references.citation: Cold Spring Harb Symp Quant Biol. 1998;63:609-20
references.pmid: 10384326
references.citation: J Immunol. 2002 Dec 15;169(12):6668-72
references.pmid: 12471095
references.citation: J Virol. 2002 Jun;76(11):5532-9
references.pmid: 11991981
references.citation: Biochem J. 2008 Jan 1;409(1):275-87
references.pmid: 17868034
references.citation: Immunopharmacology. 1996 Aug;34(1):27-37
references.pmid: 8880223
references.citation: Mol Pharmacol. 2007 Oct;72(4):868-75
references.pmid: 17615244
references.citation: Eur Neuropsychopharmacol. 2007 Dec;17(12):774-80
references.pmid: 17499975
references.citation: Proc Natl Acad Sci U S A. 1995 Dec 5;92(25):11657-61
references.pmid: 8524823
references.citation: J Immunol. 2006 Nov 1;177(9):6301-7
references.pmid: 17056560
references.citation: Science. 2003 May 16;300(5622):1148-51
references.pmid: 12702806
references.citation: Nat Immunol. 2004 Jul;5(7):730-7
references.pmid: 15208624
references.citation: Cancer Res. 2004 Mar 1;64(5):1861-6
references.pmid: 14996750
references.citation: Curr Opin Pharmacol. 2008 Feb;8(1):8-13
references.pmid: 17974478
references.citation: J Immunol. 2008 Oct 15;181(8):5522-9
references.pmid: 18832709
references.citation: J Virol. 2008 Apr;82(7):3500-8
references.pmid: 18216110
references.citation: Curr Opin Pharmacol. 2003 Aug;3(4):396-403
references.pmid: 12901949
references.citation: Nature. 2006 May 4;441(7089):101-5
references.pmid: 16625202
references.citation: J Immunol. 2008 Feb 1;180(3):1818-25
references.pmid: 18209079
references.citation: Nat Immunol. 2006 Feb;7(2):131-7
references.pmid: 16424890
references.citation: Nat Biotechnol. 2006 Feb;24(2):167-75
references.pmid: 16465162
references.citation: Cytokine Growth Factor Rev. 2005 Feb;16(1):1-14
references.pmid: 15733829
references.citation: Nat Struct Mol Biol. 2004 Nov;11(11):1060-7
references.pmid: 15502848