o-Vanillin binds covalently to MAL/TIRAP Lys-210 but independently inhibits TLR2

Md Habibur Rahaman; Sara J Thygesen; Michael J Maxwell; Hyoyoung Kim; Prerna Mudai; Jeffrey D Nanson; Xinying Jia; Parimala R Vajjhala; Andrew Hedger; Irina Vetter; Thomas Haselhorst; Avril A B Robertson; Brian Dymock; Thomas Ve; Mehdi Mobli; Katryn J Stacey; Bostjan Kobe

doi:10.1080/14756366.2024.2313055

o-Vanillin binds covalently to MAL/TIRAP Lys-210 but independently inhibits TLR2

J Enzyme Inhib Med Chem. 2024 Dec;39(1):2313055. doi: 10.1080/14756366.2024.2313055. Epub 2024 Feb 28.

Authors

Md Habibur Rahaman^{1

2

3}, Sara J Thygesen¹, Michael J Maxwell⁴, Hyoyoung Kim¹, Prerna Mudai¹, Jeffrey D Nanson^{1

2

3}, Xinying Jia⁴, Parimala R Vajjhala¹, Andrew Hedger^{1

2

3}, Irina Vetter^{3

5}, Thomas Haselhorst⁶, Avril A B Robertson^{1

3}, Brian Dymock⁷, Thomas Ve⁶, Mehdi Mobli⁴, Katryn J Stacey^{1

2}, Bostjan Kobe^{1

2

3}

Affiliations

¹ School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Australia.
² Australian Infectious Diseases Research Centre, University of Queensland, Brisbane, Australia.
³ Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia.
⁴ Centre for Advanced Imaging, University of Queensland, Brisbane, Australia.
⁵ School of Pharmacy, University of Queensland, Brisbane, Australia.
⁶ Institute for Glycomics, Griffith University, Southport, Australia.
⁷ Queensland Emory Drug Discovery Initiative, University of Queensland, Brisbane, Australia.

Abstract

Toll-like receptor (TLR) innate immunity signalling protects against pathogens, but excessive or prolonged signalling contributes to a range of inflammatory conditions. Structural information on the TLR cytoplasmic TIR (Toll/interleukin-1 receptor) domains and the downstream adaptor proteins can help us develop inhibitors targeting this pathway. The small molecule o-vanillin has previously been reported as an inhibitor of TLR2 signalling. To study its mechanism of action, we tested its binding to the TIR domain of the TLR adaptor MAL/TIRAP (MAL^TIR). We show that o-vanillin binds to MAL^TIR and inhibits its higher-order assembly in vitro. Using NMR approaches, we show that o-vanillin forms a covalent bond with lysine 210 of MAL. We confirm in mouse and human cells that o-vanillin inhibits TLR2 but not TLR4 signalling, independently of MAL, suggesting it may covalently modify TLR2 signalling complexes directly. Reactive aldehyde-containing small molecules such as o-vanillin may target multiple proteins in the cell.

Keywords: Covalent modification; MyD88 adaptor-like (MAL); Toll-like receptor (TLR); Toll/interleukin-1 receptor domain-containing adaptor protein (TIRAP); nuclear magnetic resonance (NMR); o-vanillin.

MeSH terms

Animals
Benzaldehydes*
Humans
Lysine*
Membrane Glycoproteins / metabolism
Mice
Myeloid Differentiation Factor 88 / metabolism
Receptors, Interleukin-1 / metabolism
Toll-Like Receptor 2* / metabolism
Toll-Like Receptor 4 / metabolism
Toll-Like Receptors / metabolism

Substances

Toll-Like Receptor 2
2-vanillin
Lysine
Toll-Like Receptor 4
Myeloid Differentiation Factor 88
Toll-Like Receptors
TLR2 protein, human
TIRAP protein, human
Membrane Glycoproteins
Receptors, Interleukin-1
TIRAP protein, mouse
Benzaldehydes

Grants and funding

The work was supported by the National Health and Medical Research Council (grants 1196590 and 1108859 to T.V., 1160570 to B.K., K.J.S, M.M. and T.V., 2003688 to K.J.S., and 2025931 to B.K.) and the Australian Research Council Future Fellowship (FT200100572) to T.V. and Laureate Fellowship (FL180100109) to B.K.