Research ResourceInnate Immunity

Identification of Toll-like receptor signaling inhibitors based on selective activation of hierarchically acting signaling proteins

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Sci. Signal.  14 Aug 2018:
Vol. 11, Issue 543, eaaq1077
DOI: 10.1126/scisignal.aaq1077

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Developing TLR inhibitors

Toll-like receptors (TLRs) are critical initiators of the inflammatory immune response in bacterial sepsis and other inflammatory diseases. To identify specific inhibitors of TLR signaling, Ippagunta et al. developed a stepwise phenotypic screen based on the chemically induced dimerization of TLR signaling intermediates. One compound specifically inhibited TLR signaling and prevented oligomerization of the TLR adaptor protein MyD88. Pretreatment of mice with an analog of this compound prevented bacterial toxin–induced inflammation, thus validating this strategy for the development of TLR-specific inhibitors.

Abstract

Toll-like receptors (TLRs) recognize various pathogen- and host tissue–derived molecules and initiate inflammatory immune responses. Exaggerated or prolonged TLR activation, however, can lead to etiologically diverse diseases, such as bacterial sepsis, metabolic and autoimmune diseases, or stroke. Despite the apparent medical need, no small-molecule drugs against TLR pathways are clinically available. This may be because of the complex signaling mechanisms of TLRs, which are governed by a series of protein-protein interactions initiated by Toll/interleukin-1 receptor homology domains (TIR) found in TLRs and the cytoplasmic adaptor proteins TIRAP and MyD88. Oligomerization of TLRs with MyD88 or TIRAP leads to the recruitment of members of the IRAK family of kinases and the E3 ubiquitin ligase TRAF6. We developed a phenotypic drug screening system based on the inducible homodimerization of either TIRAP, MyD88, or TRAF6, that ranked hits according to their hierarchy of action. From a bioactive compound library, we identified methyl-piperidino-pyrazole (MPP) as a TLR-specific inhibitor. Structure-activity relationship analysis, quantitative proteomics, protein-protein interaction assays, and cellular thermal shift assays suggested that MPP targets the TIR domain of MyD88. Chemical evolution of the original MPP scaffold generated compounds with selectivity for distinct TLRs that interfered with specific TIR interactions. Administration of an MPP analog to mice protected them from TLR4-dependent inflammation. These results validate this phenotypic screening approach and suggest that the MPP scaffold could serve as a starting point for the development of anti-inflammatory drugs.

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