Omeprazole activation of CD4+ and CD8+ T-cells through off-target covalent modification of cellular proteins

Targeted covalent inhibition of protein function is increasingly used as a therapeutic mode of action; however, there is a need to characterize off-target binding interactions and to understand whether this represents an immunological risk. Given that the proton-pump inhibitor omeprazole exerts its mechanism of action through covalent inhibition, it serves as an ideal model to investigate the relationship between off-target protein binding and T-cell activation. Binding of omeprazole, omeprazole metabolites, and alternative proton-pump inhibitors to antigen-presenting cells and GST-pi was characterized by mass spectrometry. Omeprazole-responsive clones were generated and assessed in terms of cytokine secretion, pathways of T-cell activation, and crossreactivity with omeprazole metabolites, alternative proton-pump inhibitors, and unrelated drugs. Omeprazole stimulated CD4+ and CD8+ T-cell clones to proliferate and secrete cytokines and cytolytic molecules. HLA-restricted T-cell activation was dependent on processing of omeprazole protein adducts by antigen-presenting cells. Omeprazole-modified CYS-containing peptides derived from 36 off-target proteins were detected within antigen-presenting cells. Omeprazole metabolites and alternative protein pump inhibitors that form protein adducts also activated omeprazole-responsive T-cells. In conclusion, T-cells were activated with omeprazole via a hapten mechanism and exhibited considerable promiscuity to metabolites and structurally related drugs of the same pharmacological class. Similar off-target binding interactions may be a relevant concern for the increasing number of covalent inhibitor drugs receiving regulatory approval.