E6446 dihydrochloride is a novel synthetic antagonist for nucleic acid-sensing TLRs; potently suppressed DNA stimulation of HEK:TLR9 cells with IC50 of 10 nM.

TLR9:10 nM.

E6446 potently inhibited IL-6 production induced by CpG2216 but was ineffective against induction by the TLR3 ligand poly inosine-cytosine. E6446 showed a modest but consistent superiority over AT791, and both were significantly more potent than hydroxychloroquine. Significantly, 250 nM and 1.25 μM E6446 completely suppressed all of the CpG oligo-induced changes in gene expression [1]. E6446 specifically inhibited TLR9 activation with CpG ODN 2006, in the range of 0.01–0.03 μM. A 100-fold higher concentration (2–8 μM) of E6446 was required to inhibit TLR7/8 activated by the imidazoquinoline compound R848 [2].

To test their activity in vivo, mice were orally dosed with 20 mg/kg of AT791 or E6446 and 18 hours later were challenged with 60 μg CpG1668 oligonucleotide injected subcutaneously. CpG1668-induced IL-6 production was inhibited ~50% by AT791 and almost completely by E6446 [1]. therapy with E6446 diminished the activation of TLR9 and prevented the exacerbated cytokine response observed during acute Plasmodium infection [2].