Molecular interactions of small molecule inhibitors targeting cytoplasmic TIR domains.

Abstract

Abstract Regulation of Toll-like receptor (TLR) signaling using small molecule agonists and antagonists have been widely sought after for controlling inflmation and disease. Most studies have focused on therapeutic targeting of TLR extracellular ligand binding domains. Recently, Computer-aided drug design (CADD) screens specifically targeting intracellular Toll-Interleukin-1 receptor resistance domains have identified and functionally characterized several small molecule and peptide inhibitors which protect against lethality in animal models of infection, inflammation and disease. We have sought to structurally characterize the molecular interactions of CADD derived small molecule inhibitors with respective TLR-TIR domains by determining the X-ray co-crystal structures of TLR2 –TIR domain in the presence of C29 and its substructure O-vanillin. Positive electron density is observed near the BB loop and residue Ile 685 of native TLR2-TIR in the presence of the small molecule inhibitor (C29) exhibit compared with the native apo form of this structure. However, the presence of (S-(DIMETHYLARSENIC) CYSTEINE) in this crystallization condition complicate analysis. To rule out the effects of DMSO and (S-(DIMETHYLARSENIC) CYSTEINE) we sought to examine small molecule inhibitor C29L substructure (o-Vanillin) in a second crystal form of TLR2 and which contains a more simplified crystallization condition grown in the presence of 1mM of C29L. This co-crystal exhibits positive electron density located in an around the BB loop compared with apo forms of this structure. Additionally, in this recent structure form we observe additional DD loop density previously not defined in the originally reported crystal form.