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)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; SanofiAventis, 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.

Therapeutic effect of hydroxychloroquine on colorectal carcinogenesis in experimental murine colitis

“…It has been reported that TLR7 and TLR9 ligands induced inflammations are inhibited by CQ/HCQ in macrophages [48,49]. CQ inhibits TLR3-induced biological response and TLR2-mediated antigen cross-presentation in immune cells [50,51].…”

Targeting downstream transcription factors and epigenetic modifications following Toll-like receptor 7/8 ligation to forestall tissue injury in anti-Ro60 associated heart block

“…The mechanism of the latter was recently challenged in a study demonstrating that the inhibitory effect of HCQ is secondary to its direct binding to nucleic acids when concentrated within the endosome, thereby preventing ligands from binding to TLRs [32,33]. …”

Translating nucleic acid-sensing pathways into therapies

“…In addition, recombinant DNA and RNA sensor proteins allow for direct binding assays to differentiate receptor-binding compounds from compounds that affect DNA-and RNAsensing pathways by a receptor-independent mode of action. In a recent example of TLR9 antagonists, suppressive activity was shown to depend on interaction of TLR9 antagonists with DNA and accumulation in endosomes 117 .…”