Demonstration of elevated levels of active cathepsin S in dextran sulfate sodium colitis using a new activatable probe.

BACKGROUND: Proteases play a major role in inflammatory diseases of the gastrointestinal tract. Activatable probes are a major technological advance, enabling sensitive detection of active proteases in tissue samples. Our aim was to synthesize an activatable probe for cathepsin S and validate its use in a mouse model of colitis. METHODS: We designed and synthesized a new fluorescent activatable probe, NB200, for the detection of active cathepsin S. Colitis was induced in C57BL/6 mice by the administration of 3% dextran sulfate sodium (DSS). Homogenized mouse colons, with or without the addition of the specific cathepsin S inhibitor MV026031, were incubated with NB200 in a fluorescent plate reader. KEY RESULTS: NB200 selectively detected purified cathepsin S and not other common inflammatory proteases. Homogenates of colon from mice with DSS colitis induced a significant fluorescent increase when compared to control animals (control vs DSS: p < 0.05 at 200 min and p < 0.01 at 220-240 min), indicating cathepsin S activation. The cathepsin S inhibitor abolished this increase in fluorescence (DSS vs DSS + MV026031: p < 0.05 at 140 min, p < 0.01 at 180 min, p < 0.001 at 200-240 min), which confirms cathepsin S activation. Cathepsin S activity correlated with the disease activity index (Spearman r = 0.77, p = 0.017). CONCLUSIONS & INFERENCES: Our investigation has demonstrated the utility of activatable probes for detecting protease activity in intestinal inflammation. Panels of such probes may allow 'signature' protease profiles to be established for a range of inflammatory diseases and disorders.

Active-site directed irreversible inhibition of diamine oxidase by a homologous series of aziridinylalkylamines.

Three electrophilic homologous aminoalkylaziridine analogues of putrescine, cadaverine, and 1,3-diaminopropane were synthesized and found to represent a mechanistically distinct class of irreversible inhibitors of diamine oxidase. The putrescine analogue, N-(4-aminobutyl)aziridine gave the lowest calculated IC50 value, whereas N-(3-aminopropyl)aziridine, an analogue of the poorest substrate of the series, showed the highest IC50. The findings suggest that the aziridinylalkylamines tested are site-directed agents that form irreversible complexes at the active site of diamine oxidase. Affinity of the inhibitors for the active site appeared to be dependent on alkyl chain length, suggesting that binding promotes the reactivity of the aziridinyl group.