Fluorescent diphenylphosphonate-based probes for detection of serine protease activity during inflammation

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Abstract

Activity-based probes are small molecules that covalently bind to the active site of a protease in an activity-dependent manner. We synthesized and characterized two fluorescent activity-based probes that target serine proteases with trypsin-like or elastase-like activity. We assessed the selectivity and potency of these probes against recombinant enzymes and demonstrated that while they are efficacious at labeling active proteases in complex protein mixtures in vitro, they are less valuable for in vivo studies. We used these probes to evaluate serine protease activity in two mouse models of acute inflammation, including pancreatitis and colitis. As anticipated, the activity of trypsin-like proteases was increased during pancreatitis. Levels of elastase-like proteases were low in pancreatic lysates and colonic luminal fluids, whether healthy or inflamed. Exogenously added recombinant neutrophil elastase was inhibited upon incubation with these samples, an effect that was augmented in inflamed samples compared to controls. These data suggest that endogenous inhibitors and elastase-degrading proteases are upregulated during inflammation.

Section snippets

Labeling proteases with ABPs

For the ABP dose curve experiment, recombinant proteases were diluted in 20 μL of phosphate-buffered saline: trypsin (100 ng), neutrophil elastase (1 μg), proteinase-3 (1 μg). The indicated ABP was added from 100x DMSO stocks to yield final concentrations of 0, 0.1, 0.5, and 1 μM with 1% DMSO. Samples were incubated for 30 min at 37 °C and then solubilized with 4× sample buffer (40% glycerol, 200 mM Tris-Cl [pH 6.8], 8% SDS, 0.04% bromophenol blue, 5% beta-mercaptoethanol). Proteins were resolved on a

Immunoprecipitation

Probe-labeled lysates were divided into input and immunoprecipitation (IP) samples (100 μg each). The pulldown sample was diluted in 500 μL IP buffer (PBS [pH 7.4], 1 mM EDTA, 0.5% NP-40) along with 5 μL sheep anti-mouse neutrophil elastase/ELA2 antibody (R&D). Protein A/G beads (40 μl slurry) were washed with IP buffer and then added to the sample. Tubes were rocked overnight at 4 °C. Beads were washed four times with IP buffer and once with 0.9% sodium chloride. After the last wash, all liquid was

Acute pancreatitis model

All animal experiments were approved by the Animal Ethics Committee of Monash University. Eight-week old male C57BL/6 J mice were given 12 hourly injections of caerulein (50 μg/kg in PBS) or PBS.12 After the ninth injection, the indicated ABP or vehicle control was administered intravenously (20 nmol dissolved in 100 μl 20% DMSO/PBS). Five hours later, pancreata were removed and imaged for Cy5 fluorescence using an IVIS Spectrum (Perkin Elmer). Radiance efficiency was determined using Living Image

Acute colitis model

Eight-week old male C57BL/6 J mice were administered 3% dextran sodium sulfate in the drinking water for 7 days. Colons were excised and flushed with 1 mL cold PBS. Supernatants were cleared by centrifugation. Total protein (50 μg) was labeled with Cy5-PK-DPP or Cy5-V-DPP and analyzed by fluorescent SDS-PAGE as above. Alternatively, elastase activity was measured with the fluorogenic substrate probe MeOSucc-Ala-Ala-Pro-Val-7-amino-4-methylcoumarin (Bachem, AAPV-AMC). Protein (50 μg) was incubated in

Cy5-V-DPP stability test

Recombinant neutrophil elastase (rNE; 1 or 10 μg as indicated) was aliquoted alone or spiked into lysates prepared from normal or inflamed pancreata or luminal fluids (100 μg total protein) followed by immediate addition of Cy5-V-DPP for 30 min at 37 °C or addition after 10 min. All samples were then analyzed using fluorescent SDS-PAGE as above and then analyzed for total elastase levels by western blot (R&D AF4517; 1:1000) and donkey anti-sheep-IR800 (LiCOR; 1:10,000). Blots were scanned on an

Statistics

All experiments were performed in triplicate using at least 3 biological replicates. Data are reported as means ± SEM. Statistical significance was determined by comparing 2 groups using the Student’s t test, and p values of less than 0.05 were considered significant.

Acknowledgements

LEM is supported by an Early Career Fellowship from the National Health and Medical Research Council (NHMRC, Australia, GNT1091636). BG is the recipient of an Australian Research Council (ARC) Future Fellowship (FT130100838). NB is supported by NHMRC and ARC Centre of Excellence in Convergent Bio-Nano Science and Technology. Work in the authors’ laboratory is funded in part by Takeda Pharmaceuticals Inc.

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