Poster Presentation The 47th Lorne Conference on Protein Structure and Function 2022

Illuminating the chemical space for MR1-restricted ligands: a binding assay using fluorescence polarisation (#106)

Carl J. H. Wang 1 , Jeffrey Y. W. Mak 2 , Ligong Liu 2 , David P. Fairlie 2 , Jamie Rossjohn 1 3 , Jerome Le Nours 1
  1. Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia
  2. Institute for Molecular Bioscience, The University of Queensland, Brisband, Queensland, Australia
  3. Institute of Infection and Immunity, Cardiff University, Cardiff, Wales, United Kingdom

Studies into T-cell-mediated immunity have largely been focused on understanding peptide presentation by Major Histocompatibility Complex (MHC) and their recognition by αβ T-cell receptors (αβ TCRs). By contrast, our understanding of non-peptide T-cell-mediated immunity in humans is limited. Mucosal-associated Invariant T-cells (MAIT cells) represent a significant population of T-cells in mucosal tissues and peripheral blood, and play a critical role in early immune responses to microbial infection.

 

MAIT cells recognise MHC class I-related (MR1) presenting small microbial vitamin B metabolites. The two major metabolites are pterin-based ligand 6-formylpterin (6-FP), a photodegradation product of folic acid (vitamin B9) (1); and pyrimidine-based ligand 5-OP-RU, being derived from microbial biosynthesis of riboflavin (vitamin B2) (2). The relative plasticity of the MR1 binding groove has recently led to the exploration of chemical space for novel MR1-restricted antigens (3). However, there is currently no method to quantify the affinity of a ligand bound to MR1. In addition, the current pipeline from initial hit to structural evidence is highly time- and resource-intensive.

 

Here, we have developed an MR1 binding assay using fluorescence polarisation (FP) technology that is able to quantify the affinity of MR1 ligands as well as provide a facile method of screening for novel ligands. We have demonstrated that the FP competitive binding assay is a suitable tool for quantifying the IC50 for MR1-restricted ligands in the nM to mM range, with affinity of the ligand correlating well with the number of polar interactions between MR1 and ligand. We have also determined the structure of a novel dietary antigen, ethylvanillin, bound to MR1. Through FACS, we were able to determine that ethylvanillin is an inhibitory molecule that can upregulate MR1 on the surface of cells. We have demonstrated that the FP competitive binding assay greatly accelerates the rate at which novel MR1 ligands may be identified and from these results, this developed tool will greatly contribute to both the chemical space of MR1-restricted ligands and to our knowledge on the biology of the MAIT-MR1 axis.

  1. Kjer-Nielsen, L., Patel, O., Corbett, A. J., Le Nours, J., Meehan, B., Liu, L., Bhati, M., Chen, Z., Kostenko, L., Reantragoon, R., Williamson, N. A., Purcell, A. W., Dudek, N. L., McConville, M. J., O’Hair, R. A. J., Khairallah, G. N., Godfrey, D. I., Fairlie, D. P., Rossjohn, J., and McCluskey, J. (2012) MR1 presents microbial vitamin B metabolites to MAIT cells. Nature 491, 717
  2. Corbett, A., Eckle, S., Birkinshaw, R., Liu, L., Patel, O., Mahony, J., Chen, Z., Reantragoon, R., Meehan, B., Cao, H., Williamson, N., Strugnell, R., Van Sinderen, D., Mak, J., Fairlie, D., Kjer-Nielsen, L., Rossjohn, J., and McCluskey, J. (2014) T-cell activation by transitory neo-antigens derived from distinct microbial pathways. Nature 509, 361-365
  3. Keller, A. N., Eckle, S. B. G., Xu, W., Liu, L., Hughes, V. A., Mak, J. Y. W., Meehan, B. S., Pediongco, T., Birkinshaw, R. W., Chen, Z., Wang, H., D'Souza, C., Kjer-Nielsen, L., Gherardin, N. A., Godfrey, D. I., Kostenko, L., Corbett, A. J., Purcell, A. W., Fairlie, D. P., McCluskey, J., and Rossjohn, J. (2017) Drugs and drug-like molecules can modulate the function of mucosal-associated invariant T cells. Nature Immunology 18, 402