In vitro activity: CU-CPT-9b, an analog of CU-CPT-8m and CU-CPT-9a, is a potent and specific antagonist of TLR8 (Toll-like receptor 8) with IC50 of 0.7 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. ITC experiments have confirmed the strong binding of CU-CPT-9b with a Kdof 21 nM. It is shown that CU-CPT-9b binds to the inactive TLR8 dimer in a similar way to CU-CPT8m. CU-CPT9b utilizes hydrogen bonds with G351 and V520*, which are conserved among TLR8/antagonist structures. Additionally, CU-CPT-9b forms water-mediated contacts with S516* and Q519*, which are not observed in TLR8/CU-CPT8m structure, suggesting that the enhanced potency of CU-CPT-9b derives from the new interactions with these polar residues. The orientation of Y567* also changes to facilitate van der Waals interactions with CU-CPT-9b as compared to TLR8/CU-CPT8m..

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.