Chemical Tools to Image the Activity of PAR-Cleaving Proteases

Abstract

Protease-activated receptors (PARs) comprise a family of four G protein-coupled receptors (GPCRs) that have broad functions in health and disease. Unlike most GPCRs, PARs are uniquely activated by proteolytic cleavage of their extracellular N termini. To fully understand PAR activation and function in vivo, it is critical to also study the proteases that activate them. As proteases are heavily regulated at the post-translational level, measures of total protease abundance have limited utility. Measures of protease activity are instead required to inform their function. This review will introduce several classes of chemical probes that have been developed to measure the activation of PAR-cleaving proteases. Their strengths, weaknesses, and applications will be discussed, especially as applied to image protease activity at the whole organism, tissue, and cellular level.

Figure 1

Mechanisms of common substrate-based probes and activity-based probes. a) Internally quenched fluorescent SBP, containing a peptide flanked by a fluorophore–quencher pair. b) Fluorogenic SBP containing a 7-amino-4-carbamoylmethyl coumarin (ACC) fluorophore that fluoresces only upon cleavage of the peptide at the P1 site. c) FRET SBP, in which a protease cleavage site is flanked by a donor and acceptor fluorophore. d) Hairpin FRET, in which a protease cleavage site is flanked by positively and negatively charged peptides. Upon extracellular cleavage, the FRET signal is reduced, and the positively charged cell-penetrating-peptide is able to accumulate in cells. e) An example of a nonquenched fluorescent ABP bearing a diphenylphosphonate (DPP) warhead. This probe is always fluorescent so requires washout of unbound probe for imaging applications. f) An example of a fluorescently quenched ABP bearing an acyloxymethyl ketone (AOMK) warhead. This probe fluoresces only upon release of the quenching group upon nucleophilic attack by the protease. Note as SBPs do not inhibit the protease, many probe molecules can be cleaved by the same protease allowing amplification, while ABPs inhibit the protease (1:1 reaction).

Figure 2

Top-down imaging of protease activity with activity-based probes. Many fluorescent ABPs and SPBs can be administered in vivo for whole-organism, noninvasive imaging applications. Tissues can be excised for ex vivo imaging and sectioned for microscopy to examine protease activity at the cellular level. Labeling with covalent ABPs can also be observed at the protein level by in-gel fluorescence, to verify specificity of the probe in the target tissue. This is an example of imaging with the fluorescently quenched legumain probe, LE28, in a mouse model of acute pancreatitis. These images were obtained from the same mouse: whole animal, pancreas tissue, pancreas section (magenta = LE28; blue = DAPI) and in-gel fluorescence of resolved pancreas lysate.