ETI60 is an orally active selective TLR inhibitor that targets the nucleoside-binding sites I of TLR7 (IC50 = 0.68 μM) and TLR9 (IC50 = 0.12 μM) without affecting surface TLRs (including TLR1/TLR2, TLR2/TLR6, TLR4, and TLR5). It demonstrates nanomolar activity in cell-based, biophysical, and in vivo assays, effectively inhibiting endosomal TLR-mediated pro-inflammatory signaling. ETI60 modulates the expression of genes related to inflammation and significantly improves symptoms in mouse models of psoriasis and systemic lupus erythematosus (SLE). ETI60 is applicable in studies related to autoimmune and inflammatory diseases.

TLR7:0.68 μM

ETI60 effectively inhibits TNF-α production in mouse macrophages (RAW 264.7) and human B lymphoblastoid (Daudi) cell lines induced by TLR agonists, such as Imiquimod (IMQ) for TLR7 and ODN2395 for TLR9, within the range of 0-200 μM and 4-24 hours, without causing cytotoxic effects. ETI60 also inhibits TLR3, TLR7, and TLR9 with IC50 values between 20 and 300 nM, and TLR8 with an IC50 of approximately 2 μM. It suppresses the activity of TLR3, TLR7, and TLR8 in a concentration-dependent manner within the range of 31.2 nM to 10 μM. Additionally, ETI60 (5-10 μM, 20 minutes to 8 hours) reduces phosphorylation of MAPKs (p-ERK, p-JNK, and p-p38), nuclear translocation of NF-κB p65 subunit, Iκ-Bα degradation, and expression of IRF7 in RAW 264.7 cells induced by IMQ or ODN2395. Finally, ETI60 (10 μM, 2-4 hours) attenuates the upregulation of multiple IMQ-induced inflammation-related genes, including IL1-β, CXCL2, IL18RAP, TNF, PDCD1, NLRP3, NFKBIZ, CCL3, CCL4, KDM6B, ZC3H12A, and PTGS2.

ETI60, administered orally at 60 mg/kg once daily from day 2 to day 5, can alleviate IMQ-induced psoriasis in mice. When given at the same dose daily from day 2 to day 9, it also improves IL-23-induced psoriasis in mice. Additionally, a dosage of ETI60 at 30 mg/kg daily for 39 days effectively alleviates symptoms in a systemic lupus erythematosus (SLE) mouse model.