Toll-like receptors (TLRs) are an integral part of the innate immune system. They serve as both extracellular and endosomal sensors for a variety of pathogen-associated molecular patterns. Inappropriate activation of the endosomal TLRs 7 and 8 has been implicated in the pathogenesis of a series of autoimmune diseases, including lupus.

Previous hit generation strategies for identification of TLR modulators relied on cellular pathway assays, in which TLR expressing cells are stimulated with TLR specific agonists to elicit cytokines. While this approach has been successfully applied for decades, validation and optimization of hits from cellular screening campaigns can be time and resource intensive. In order to demonstrate target specificity a battery of counter-assays is required and rational optimization is complicated by the complexity of the assay system.

In this presentation we describe our efforts to identify and optimize TLR 7/8 antagonists with novel assays using the recombinant endosomal TLR 8 domain. A TLR 8 binding assay was set up using a labeled antagonist as probe and applied for high-throughput screening. The validated hits could be efficiently optimized in a structure-guided manner resulting in highly potent and selective TLR 7/8 antagonists with demonstrated in vivo efficacy after oral dosing.