IC50 determination

IC₅₀ values of compounds against Mtb20SOG, β5i and β5c were determined from tests in a 96-well format as reported ²⁰. In brief, 1 μL of compound in a 3x series dilution in DMSO at concentrations from 100 μM to 0.0017 μM were spotted to the bottom of a black 96-well plate having a solid bottom. Reaction buffer (100 μL; 20 mM HEPES, 0.5 mM EDTA, pH 7.5) containing enzyme and substrate were dispensed into each well, and the plate was then spun in a simple bench top plate centrifuge at 1000x rpm for 1 min, followed by shaking on an orbital shaker for 1 min. The time course of the hydrolysis in each well was followed by recording the fluorescence of the product AMC (λ_ex 360 nm and λ_em 460 nm) on a SpectraMax M5 plate reader over 1.5–2 h. The initial reaction velocity of each well was fit to a dose-dependent inhibition equation using PRISM to determine the IC₅₀. IC₅₀ values were determined only for β5i and β5c. Inhibitory activities against β1i, β1c, β2i, and β2c were tested at 100 μM, 33.3 μM, and 11.1 μM. All compounds showed < 50% inhibition at 33.3 μM, hence, the IC₅₀ values are presented as > 33.3 μM (Table S1). Suc-LLVY-AMC was used for Mtb20SOG activity at 50 μM and for c-20S β5c at 25 μM. Ac-ANW-AMC was used for i-20S β5i activity at 15 μM. Z-LLE-AMC was used for i-20S β1i and c-20S β1c activity at 50 μM, and Z-VLR-AMC was used for i-20S β1i and c-20S β1c activity at 50 μM. Final concentrations of Mtb20S, β5i and β5c were 2 nM, 0.4 nM and 0.2 nM, respectively. Sodium dodecyl sulfate was used as activator at 0.02% for human β5i, β5c, β1i and β1c. PA28a was used as activator at 4x equivalent of β2i and β2c.