title: Novel small molecule inhibitors of TLR7 and TLR9: mechanism of action and efficacy in vivo.
abstract: The discovery that circulating nucleic acid-containing complexes in the serum of autoimmune lupus patients can stimulate B cells and plasmacytoid dendritic cells via Toll-like receptors 7 and 9 suggested that agents that block these receptors might be useful therapeutics. We identified two compounds, AT791 {3-[4-(6-(3-(dimethylamino)propoxy)benzo[d]oxazol-2-yl)phenoxy]-N,N-dimethylpropan-1-amine} and E6446 {6-[3-(pyrrolidin-1-yl)propoxy)-2-(4-(3-(pyrrolidin-1-yl)propoxy)phenyl]benzo[d]oxazole}, that inhibit Toll-like receptor (TLR)7 and 9 signaling in a variety of human and mouse cell types and inhibit DNA-TLR9 interaction in vitro. When administered to mice, these compounds suppress responses to challenge doses of cytidine-phosphate-guanidine (CpG)-containing DNA, which stimulates TLR9. When given chronically in spontaneous mouse lupus models, E6446 slowed development of circulating antinuclear antibodies and had a modest effect on anti-double-stranded DNA titers but showed no observable impact on proteinuria or mortality. We discovered that the ability of AT791 and E6446 to inhibit TLR7 and 9 signaling depends on two properties: weak interaction with nucleic acids and high accumulation in the intracellular acidic compartments where TLR7 and 9 reside. Binding of the compounds to DNA prevents DNA-TLR9 interaction in vitro and modulates signaling in vivo. Our data also confirm an earlier report that this same mechanism may explain inhibition of TLR7 and 9 signaling by hydroxychloroquine (Plaquenil; Sanofi-Aventis, Bridgewater, NJ), a drug commonly prescribed to treat lupus. Thus, very different structural classes of molecules can inhibit endosomal TLRs by essentially identical mechanisms of action, suggesting a general mechanism for targeting this group of TLRs.
authors.affiliation: Eisai, Inc., Andover, Massachusetts (M.L., W.Z., M.S., H.H., J.R., M.G., H.Y., C.S., Y.Z., Y.S., C.L., D.L., C.R., J.C., N.C.T., M.Y., F.G., S.T.I.); Department of Infectious Diseases and Immunology, University of Massachusetts, Worcester, Massachusetts (E.L.); Institute of Innate Immunity, University of Bonn, Germany (E.L.); Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital, Charlestown, Massachusetts (T.R.M.); and Center for Systems Biology, Harvard Medical School, Charlestown, Massachusetts (T.R.M.).
authors.forename: Marc
authors.initials: M
authors.lastname: Lamphier
authors.forename: Wanjun
authors.initials: W
authors.lastname: Zheng
authors.forename: Eicke
authors.initials: E
authors.lastname: Latz
authors.forename: Mark
authors.initials: M
authors.lastname: Spyvee
authors.forename: Hans
authors.initials: H
authors.lastname: Hansen
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Rose
authors.forename: Melinda
authors.initials: M
authors.lastname: Genest
authors.forename: Hua
authors.initials: H
authors.lastname: Yang
authors.forename: Christina
authors.initials: C
authors.lastname: Shaffer
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhao
authors.forename: Yongchun
authors.initials: Y
authors.lastname: Shen
authors.forename: Carrie
authors.initials: C
authors.lastname: Liu
authors.forename: Diana
authors.initials: D
authors.lastname: Liu
authors.forename: Thorsten R
authors.initials: TR
authors.lastname: Mempel
authors.forename: Christopher
authors.initials: C
authors.lastname: Rowbottom
authors.forename: Jesse
authors.initials: J
authors.lastname: Chow
authors.forename: Natalie C
authors.initials: NC
authors.lastname: Twine
authors.forename: Melvin
authors.initials: M
authors.lastname: Yu
authors.forename: Fabian
authors.initials: F
authors.lastname: Gusovsky
authors.forename: Sally T
authors.initials: ST
authors.lastname: Ishizaka
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D004317:Doxorubicin / Q000494:pharmacology; D048848:MAP Kinase Kinase Kinase 5 / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D018384:Oxidative Stress / Q000187:drug effects / Q000235:genetics; D050199:Podocytes / Q000187:drug effects / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D054852:Small Molecule Libraries / Q000493:pharmacokinetics*; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D051199:Toll-Like Receptor 7 / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D051217:Toll-Like Receptor 9 / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D017382:Reactive Oxygen Species; D054852:Small Molecule Libraries; C477038:Tlr7 protein, mouse; C495370:Tlr9 protein, mouse; D051199:Toll-Like Receptor 7; D051217:Toll-Like Receptor 9; C445672:Txnip protein, mouse; D013879:Thioredoxins; D004317:Doxorubicin; D048051:p38 Mitogen-Activated Protein Kinases; D048848:MAP Kinase Kinase Kinase 5; C482859:Map3k5 protein, mouse; D055372:AMP-Activated Protein Kinases