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

Abstract 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 MALTIR and MyD88TIR, 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. Competing Interest Statement The Korea Institute of Science and Technology has filed a patent on the peptides identified in this study. The patent mentions M.-H.S, W.L, K.P, S.H.J, Y.L, and M.-I.K as the inventors. The other authors declare no competing interests.