In vitro activity: CU-CPT-9a, an analog of CU-CPT-8m, is a potent and specific antagonist of TLR8 (Toll-like receptor 8) with IC50 of 0.5 nM. Endosomal Toll-like receptors (TLR3, TLR7, TLR8, and TLR9) are highly analogous sensors for various viral or bacterial RNA and DNA molecular patterns. Nonetheless, few small molecules can selectively modulate these TLRs. The elevation of the downstream protein levels induced by R848 can be reversed by CU-CPT-9a in a dose-dependent manner. By contrast, the expression of TRIF and IRF3 (cytoplasmic and nuclear) are only responsive to TLR4 and TLR3, independent of TLR837. The expression levels of TRIF and IRF3 do not show significant change in THP-1 cells upon treatment of R848, nor do they change with the treatment of CU-CPT-9a. CU-CPT8m and CU-CPT-9a both significantly suppress the TNF-α level in a dose-dependent manner, which is in agreement with previous reports of TLR8 involvement in these autoimmune diseases.

Kinase Assay: The selectivity of compounds against the TLR family was examined in HEK-Blue cells overexpressing a specific TLR and accessory proteins. The assay was performed in the same manner as “SEAP reporter assay”, except that polyriboinosinic:polyribocytidylic acid (poly(I:C)) (5 μg/mL), LPS (lipopolysaccharide) (20 ng/mL), Pam3CSK4 (N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysineo3HCl) (100 ng/mL), Pam2CSK4 (S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysineo3CF3COOH) (100 ng/mL), Flagellin (50 ng/mL), R848 (1 μg/mL), ODN2006 (0.15 μM) were used to selectively activate HEK-Blue hTLR3, hTLR4, hTLR1/2, hTLR2/6, hTLR5, hTLR7, and hTLR9 cells, respectively.

Cell Assay: HEK-Blue TLR8 cells were plated at 3.5 × 105 cells/mL in a tissue culture treated 96-well plate in DMEM with 10% (v/v) FBS (deactivated phosphatases). Then cells were treated with 1 μg/mL R848 (Invivogen) and varying concentrations of appropriate compounds. Cells were incubated with compounds and R848 at 37 °C. After 20–24 h of incubation, 20 μL of culture media was removed and placed in a new 96-well plate. 180 μL of Quanti-Blue (Invivogen) was added to the media, and the plate was incubated at 37 °C until color change was observed (30 min–1 h). Plates were then quantified on a Beckman-Coulter DTX 880 Multimode Detector by measuring absorbance at 620 nm. Data was normalized as readout of ligand treated cells is 100% activation, and untreated cells are 0% activation.