In vitro activity: CU-CPT-8m is a potent and specific antagonist of Toll-like receptor 8 (TLR8) with an IC50 of 67 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. CU-CPT-8m is the first human TLR8-specific small-molecule antagonists via a novel inhibition mechanism. Crystal structures of two distinct TLR8-ligand complexes validated a unique binding site on the protein-protein interface of the TLR8 homodimer. Upon binding to this new site, the small-molecule ligands stabilize the preformed TLR8 dimer in its resting state, preventing activation. CU-CPT-8m is able to suppress TLR8-mediated proinflammatory signaling in various cell lines, human primary cells, and patient specimens. These results not only suggest a novel strategy for TLR inhibitor design, but also shed critical mechanistic insight into these clinically important immune receptors.

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.