BODIPY-tagged β-lactams as selective quenched activity-based probes to target human neutrophil elastase

Activity-based probes are indispensable tools for interrogating protease function, and quenched fluorescent variants enable dynamic, real-time imaging of enzymatic activity. Despite these advances, very few quenched activity-based probes (qABPs) have been reported for serine proteases, which constitute the largest and most diverse mechanistic class of proteases. β-Lactams have been extensively used to develop molecular tools and drugs designed to bind or be hydrolysed by serine-dependent bacterial enzymes. Here, we report the first monocyclic β-lactam-containing qABPs for detecting serine proteases. Both the enzyme-triggered activation mechanism and intrinsic reactivity of the probes were highly dependent on the relative position of the BODIPY-FL fluorophore and quencher moiety at the β-lactam core. qABPs displaying the most efficient turn-on mechanism were shown to selectively target human neutrophil elastase (HNE) in different human cell lysates. The most successful qABP was rapidly internalised and targeted HNE in U937 cells and human neutrophils. These results demonstrate the potential of the modular β-lactam warhead to develop turn-on probes to track neutrophil serine proteases in live cells.