Pa28α

The screening of compounds was performed at 1 μM final concentrations in the assay buffer (0.01% SDS, 50 mM Tris-HCl, 0.5 mM EDTA, pH 7.4). Stock solutions of compounds were prepared in DMSO. To 50 μL of each compound, 25 μL of 0.8 nM human iCP or human cCP (both from Boston Biochem, Inc., Cambridge, MA, USA) was added. After 30 min incubation at 37 °C, the reaction was initiated by the addition of 25 μL of 100 μM relevant fluorogenic substrate: acetyl-Nle-Pro-Nle-Asp-AMC (Ac-nLPnLD-AMC, [Bachem, Bubendorf, Switzerland]) for β1, acetyl-Pro-Ala-Leu-7-amino-4-methylcoumarin (Ac-PAL-AMC, [Boston Biochem, Inc., Cambridge, MA, USA]) for β1i, t-butyloxycarbonyl-Leu-Arg-Arg-7-amino-4-methylcoumarin (Boc-LRR-AMC, [Bachem, Bubendorf, Switzerland]) for β2 and β2i, succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC [Bachem, Bubendorf, Switzerland]) for β5 and β5i. The reaction progress was recorded on the BioTek Synergy HT microplate reader by monitoring fluorescence at 460 nm (λ_ex = 360 nm) for 90 min at 37 °C. The initial linear ranges were used to calculate the velocity and to determine the residual activity.
In the case of the β1, β1i, β2, and β2i activity inhibition determination, the assay buffer was modified; SDS was replaced with the proteasomal activator PA28α (Boston Biochem, Inc., Cambridge, MA, USA).

Residual activity measurements were performed at 10 μM final concentrations in the assay buffer (0.01% SDS, 50 mM Tris-HCl, 0.5 mM EDTA, pH 7.4). Stock solutions of the compounds were prepared in DMSO. To 50 μL of each compound in buffer, 25 μL of 0.8 nM human iCP or human cCP (both from Boston Biochem, Inc., Cambridge, MA, USA) were added. After incubation at 37 °C for 30 min, the reaction was started by adding of 25 μL of 100 μM relevant fluorogenic substrate: acetyl-Nle-Pro-Nle-Asp-AMC (Ac-nLPnLD-AMC, (Bachem, Bubendorf, Switzerland)) for β1, acetyl-Pro-Ala-Leu-7-amino-4-methylcoumarin (Ac-PAL-AMC, (Boston Biochem, Inc., Cambridge, MA, USA)) for β1i, t-butyloxycarbonyl-Leu-Arg-Arg-7-amino-4-methylcoumarin (Boc-LRR-AMC, (Bachem, Bubendorf, Switzerland)) for β2 and β2i, succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC) (Bachem, Bubendorf, Switzerland) for β5 and β5i. Reaction progress was recorded on BioTek Synergy HT microplate reader by monitoring fluorescence at 460 nm (λ_ex = 360 nm) for 90 min at 37 °C. The initial linear ranges were used to calculate velocity and determine residual activity. In the case of evaluation of β1, βi, β2, and β2i activities, the assay buffer was modified by replacing SDS with the proteasomal activator PA28α (Boston Biochem, Inc., Cambridge, MA, USA).