Development of Novel Activity-Based Probes for the Detection of Serine Proteases

Cystic Fibrosis (CF) is a genetic disease featuring a chronic cycle of inflammation and infection in the airways of sufferers. Mutations lead to altered ion transport, which in turn causes dehydrated airways and reduced mucociliary clearance which predisposes the patient to infection, resulting in a severe immune response and tissue destruction (1). Airway dehydration is primarily caused by the hyperabsorption of sodium by the epithelial sodium channel (ENaC) (2). ENaC is activated by the action of a number of predominantly trypsin-like Channel Activating Proteases (CAPs) including prostasin, matriptase and furin (3). Additional proteases known to activate ENaC include human airway trypsin (3), plasmin, neutrophil elastase and chymotrypsin (4).

Activity profiling is a valuable technique which involves the use of small inhibitory molecules called Activity-Based Probes (ABPs) which can be used to covalently label the active site of proteases and provide a range of information regarding its structure, catalytic mechanism, location and function within biological systems. The development of novel ABPs for CAPs, would enhance understanding of the role of these proteases in CF airways disease and in particular their role in ENaC activation and airway dehydration. This project investigates the application of a range of novel broad-spectrum ABPs targeting the various subclasses of serine proteases, to include those proteases involved in ENaC activation. Additionally, the application of more selective ABPs in detecting specific serine proteases is investigated.

Compounds were synthesised by Solid-Phase Peptide Synthesis (SPPS) using a standard Fmoc/tBu strategy. Kinetic evaluation of synthesised ABPs against various serine proteases was determined by fluorogenic steady-state enzyme assays. Furthermore, application of ABPs and confirmation of irreversible nature of the compounds was carried out through SDS-PAGE and electroblotting techniques.

Synthesised compounds showed potent irreversible inhibition of serine proteases within their respective targeting class (NAP855 vs Trypsin k3/Ki = 2.60 x 106 M-1 min-1, NFP849 vs Chymotrypsin k3/Ki = 1.28 x 106 M-1 min-1 and NVP800 vs Neutrophil Elastase k3/Ki = 6.41 x 104 M-1 min-1). Furthermore ABPs showed little to no cross-reactivity between classes and so display selectivity between classes. The irreversible nature of compounds was further demonstrated through labelling of proteases, followed by separation and detection via SDS-PAGE and electroblotting techniques. Targeted labelling of active proteases only, was demonstrated by failure of ABPs to detect previously inactivated proteases. Extension of the substrate recognition site within probes resulted in an increased potency and selectivity in the detection of the target proteases. Successful detection of neutrophil elastase from CF sputum samples by NVP800, demonstrated the application of compounds within biological samples and their potential use in identifying further proteases involved in ENaC activation and airway dehydration in CF patients.