Label-free flow cytometry-based enzyme inhibitor identification

Fluorescence-based methods for the identification of enzyme inhibitors are widespread, but usually require protein or ligand labelling. In this study, we present a label-free displacement assay that takes advantage of the intrinsic fluorescence of a tight binding ligand avoiding any labeling. Autodisplay-based accessibility of the target enzyme on the cell surface of Escherichia coli enabled the quantification of fluorescent ligand binding by flow cytometry. Human protein kinase CK2 was used as proof-of-concept enzyme and its ATP competitive inhibitor (E)-1,3-dichloro-6-[(4-methoxyphenylimino)methyl]dibenzo[b,d]furan-2,7-diol (compound 5) was shown to exhibit intrinsic fluorescence (λ_max(ex) = 370 nm, λ_max(em) = 585 nm). Binding of compound 5 to CK2 displaying cells was quantified via flow cytometry with linearly increasing relative fluorescence up to a concentration of 1.25 μM. The addition of the non-fluorescent CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) competed for compound 5 binding with a half maximal fluorescence reduction at 15.6 μM TBB. This new and simple binding assay provides a valuable tool for the screening of high affinity enzyme inhibitors, overcoming the limitations of fluorescent ligand labelling.