The chemical synthesis, stability and activity of MAIT cell prodrug agonists that access MR1 in recycling endosomes.

Mucosal-associated invariant T (MAIT) cells are anti-bacterial effector T cells that react to pyrimidines derived from bacte-rial riboflavin synthesis. We show here that a major challenge in MAIT cell research is that the commonly used MAIT ago-nist precursor, 5-amino-6-D-ribitylaminouracil (5-A-RU), is labile to auto-oxidation resulting in loss of biological activity. To overcome this, we report the synthesis of a 5-A-RU prodrug generated by modification of the 5-amino group with a cleava-ble valine-citrulline-p-aminobenzyl carbamate. The compound is stable in prodrug form, with the biologically active MAIT agonist precursor only released after enzymatic cleavage. Analysis of the prodrug in vitro and in vivo showed an enhanced MAIT cell activation profile compared to 5-A-RU, which was associated with preferential loading within recycling endo-somes, a route used by some natural agonists. This prodrug design therefore overcomes the difficulties associated with 5-A-RU in biological studies, and provides an...