Discovery of small-molecule inhibitors of the TLR1/TLR2 complex

Abstract

The protein complex of toll-like receptor 1 and 2 (TLR1/2) is an important regulator of innate immunity, and therefore provides an attractive target for the treatment of various immune disordres. Here we report a novel compound (CU-CPT22) that can compete with the synthetic triacylated lipoprotein (Pam₃CSK₄) binding to TLR1/2 with high inhibitory activity and specificity. Repression of downstream signaling from TNF-α and IL-1β has also been observed.

Figure 1

a) Dose-dependent inhibition of NO production in RAW 264.7 macrophage cells by CU-CPT22 and the negative control compound 6. b) Binding site prediction of CU-CPT22 (show in the stick representation) to TLR1/2 performed by Glide 5.6 program^[23]. The six-membered carbon chain fits well into the hydrophobic channel of hTLR1, having key hydrophobic interactions with Val311, Phe312, Pro315 and Val339.

Figure 2

Fluorescence anisotropy titration: a) titration of the TLR1/2 protein into the rhodamine-labeled Pam₃CSK₄ results in significant increase of fluoresence anistropy. Addition of CU-CPT22 competes with Pam₃CSK₄, resulting in lower fluoresence anistropy, demonstrating competitive binding between CU-CPT22 and Pam₃CSK₄ for TLR1/2. Data were fitted using a one-site competition model (R² > 0.98). b) Normalized binding of CU-CPT22 compared with the negative control, compound 6.

Figure 3

Specificity test for CU-CPT22 (0.5 µM) with with TLR-specific agonists used to selectively activate the respective TLRs: (1) TLR3: 15 µg/mL Poly (I:C), (2) TLR4: 10 ng/mL LPS, (3) TLR1/2: 200 ng/mL Pam₃CSK₄, (4) TLR2/6: 10 ng/mL FSL-1, and (5) TLR7: 100 nM R848 were used to selectively activate respective TLRs.

Figure 4

Downstream signaling inhibition. a) ELISA assay results showed that CU-CPT22 inhibits the TNF-α and b) IL-1β production activated by 300 ng/ml Pam₃CSK₄ in the RAW 264.7 cells.

Scheme 1

Representative synthesis for CU-CPT22: a) phosphate-citrate buffer (pH 5), 0.2 M Na₂HPO₄: 0.1 M citrate = 1:1; b) horseradish peroxidase enzyme catalyst; c) four aliquots of 3% H₂O₂, 42% overall yield.