Z fr amc

Recombinant human cathepsin K was prepared as described previously [24] . Chemical probes were obtained from the U.S. NCI/DTP Open Chemical Repository or from ChemBridge (USA). Information on their identity and origin is given in Table 1. All compounds were soluble in aqueous buffers at concentrations of at least 2 mM, except for compound 2, which was soluble up to a maximal concentration of 1 mM. Stock solutions of all compounds were prepared in DMSO. The fluorogenic substrate Z-FR↓AMC (benzyloxycarbonyl-Phe-Arg-7-amino-4-methylcoumarin) was from Bachem (Switzerland).

To evaluate the influence of Bet_v_1 ligands on cathepsin S and legumain activities, 10 nmol/L of protease was incubated with 100 µmol/L of ligand (unless otherwise stated) and 50 µmol/L of fluorogenic substrate in digestion buffer (0.1 mol/L sodium acetate pH 5.0, 0.1 mol/L sodium chloride, 5 mmol/L EDTA, and 2 mmol/L DTT), as described in the Appendix [Link], [Link]. The effect of birch pollen extract (BPE) (20‐200 µg/mL) on the cathepsin S and legumain activities was assessed in parallel. The inhibitory effect of PPE₁ was assessed by replacing DTT with 0.5 mmol/L TCEP. Activities of recombinant rat cathepsin B (provided by Dr Lukas Mach) and papain (Merck) at 10 nmol/L were assayed using Z‐FR‐AMC (Bachem) as a fluorogenic substrate.

Triton X-100, sodium acetate, DMSO, DTT (dithiothreitol) and E-64 (trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane) were purchased from Sigma-Aldrich. The Cruzipain model fluorogenic substrate Z-FR-AMC (benzyloxycarbonyl-phenylalanylarginine-4-methylcoumaryl-7-amide) was from Bachem (Bubendorf, Switzerland). Black solid bottom polystyrene Corning® NBS 384-well plates were from Sigma-Aldrich (CLS3654-100EA).

Proteolytic activities were measured in a kinetic continuous assay using the following peptidyl fluorogenic, 7-amino-4-methylcoumarin (AMC) substrates (Bachem) at a 50 µM final concentration: Z-F-R-AMC (Z, Benzyloxycarbonyl), Bz-F-V-R-AMC (Bz, Benzoyl), Z-G-P-R-AMC, P-F-R-AMC, Boc-L-R-R-AMC (Boc, t-Butyloxycarbonyl), Boc-Q-A-R-AMC, Boc-V-L-K-AMC Suc-A-A-F-AMC (Suc, Succinyl), Suc-A-A-P-F-AMC, Suc-L-Y-AMC, MeOSuc-A-A-P-V-AMC (MeOSuc, 3-Methoxysuccinyl), Z-G-G-L-AMC and Z-V-K-M-AMC. Assays were performed at 37°C in 96-well black microplates in a total volume of 100 µl. Parasite extracts (1–3 µg) or E/S products (0.05–1 µg) were pre-incubated for 10 min in 150 mM Tris-HCl, pH 8.0, containing 10 µM E64, 1 mM EDTA in the presence or absence of 0.5 mM of the serine protease inhibitors, Pefabloc SC and PMSF. E64 was included routinely in extract preparations in order to inhibit Clan CA cysteine protease activity that is present in the life-stages examined [30] (link), [39] (link), [40] (link). Hydrolysis of substrate was measured continuously using an Infinite M1000 microplate reader (Tecan) at excitation and emission wavelengths of 360 and 465 nm, respectively. All measurements were performed in triplicate and results normalized to protein concentration.