Massively parallel screening of TIR-derived peptides reveals vast TLR-targeting immunomodulatory peptides

Toll-like receptors (TLRs) are critical regulators of the immune system, and altered TLR responses lead to a variety of inflammatory diseases. Interference of intracellular TLR signaling, which is mediated by multiple Toll/interleukin-1 receptor (TIR) domains on all TLRs and TLR adapters, is an effective therapeutic strategy against immune dysregulation. Peptides that inhibit TIR-TIR interactions by fragmenting interface residues have potential as therapeutic decoys. However, a systematic method for discovering TIR-targeting moieties has been elusive, limiting exploration of the vast unsequenced space of the TIR domain family. Here, we developed a comprehensive parallel screening method to uncover novel TIR-binding peptides derived from previously unexplored surfaces on a wide range of TIR domains. We constructed a large peptide library, named TIR surfacesome, by tiling surface sequences of the large TIR domain family and screening against MAL TIR and MyD88 TIR , TIRs of two major TLR adaptor proteins, resulting in the discovery of hundreds of TIR-binding peptides. The selected peptides inhibited TLR signaling, demonstrated anti-inflammatory effects in macrophages and therapeutic potential in mouse inflammatory models. This approach may facilitate the development of TLR-targeted therapeutics.