Poster Presentation The 46th Lorne Conference on Protein Structure and Function 2021

Targeting TIR domain assemblies in TLR signalling pathways to design anti-inflammatory compounds (#107)

Md Habibur Rahaman 1 , Thomas Ve 2 , Thomas Haselhorst 2 , Mehdi Mobli 3 , Jeff Nanson 1 , Xinying Jia 3 , Parimala R. Vajjhala 1 , Sara Thygesen 1 , Kate J. Stacey 1 , Bostjan Kobe 1
  1. School of Chemistry and Molecular Biosciences, Institute for Molecular Bioscience and Australian Infectious Diseases Research Centre, University of Queensland, Brisbane, QLD 4072, Australia
  2. Institute for Glycomics, Griffith University, Southport, QLD 4222, Australia
  3. Centre for Advanced Imaging, University of Queensland, Brisbane, QLD 4072, Australia

TLRs (Toll-like receptors) are central components of mammalian innate immunity. Upon activation, their cytosolic TIR (Toll/interleukin-1 receptor) domains recruit the TIR-domain containing adaptor proteins MyD88 and MAL via TIR: TIR interactions. MAL works as a bridging adaptor for the association of MyD88 with TLR2 or TLR4, which in turn activates the transcription factor NF-kB to produce pro-inflammatory cytokines.

Over-activation of this signalosome may lead to chronic inflammatory conditions. Therefore, a broad-spectrum anti-inflammatory drug can be designed by targeting TLR2/4 signalling. TLR-antagonists designed against the extracellular domains of the receptors have had limited clinical outcomes. Targeting protein-protein interactions of the downstream adaptors provides an alternative approach.

The TIR-domain of MAL (MALTIR) forms spontaneous and reversible filaments in vitro. The cryo-EM structure of the MALTIR filament revealed intra- and inter-strand interfaces. Several interfacial residues are crucial for mediating TLR4 signalling. Although such structural information could be exploited for structure-based drug design, to our knowledge, no small molecule inhibitors of MALTIR have been reported.

We have recently identified several small molecule inhibitors of MALTIR filament. Among them, o-vanillin is the most potent lead molecule. To reveal the structure-activity relationships of o-vanillin-mediated MALTIR inhibition, the solution structure of the wild-type MALTIR was determined. Detailed HSQC and intermolecular-NOE analyses showed that o-vanillin preferentially targets the intrastrand interface of MALTIR filament. In particular, o-vanillin forms a Schiff base with the K210 of MALTIR, which is proximal to the intrastrand interface. Therefore, this interface of the MALTIR filament may be affected by o-vanillin induced side-chain orientation of K210, as predicted from the intermolecular-NOE-derived distance restraints. The aldehyde group of o-vanillin and its ortho-hydroxyl moiety were found to be indispensable for its inhibitory activity.

Overall, this study provides the first proof-of-principle that MALTIR function can be inhibited by small molecules, which can be advanced further as potential anti-inflammatories.