Calpain inhibitor

For reaction condition optimization, 200 µM SARS-CoV-2 M^pro was used. pH 6.0 buffer contains 20 mM MES, pH 6.0, 120 mM NaCl, 0.4 mM EDTA, 4 mM DTT and 20% glycerol; pH 6.5 buffer contains 20 mM HEPES, pH 6.5, 120 mM NaCl, 0.4 mM EDTA, 4 mM DTT and 20% glycerol; pH 7.0 buffer contains 20 mM HEPES, pH 7.0, 120 mM NaCl, 0.4 mM EDTA, 4 mM DTT and 20% glycerol. Upon addition of 20 µM FRET substrate, the reaction progress was monitored for 1 h. The first 15 min of reaction was used to calculate initial velocity via linear regression in Prism 5. M^pro displays the highest proteolytic activity in pH 6.5 buffer. All the following enzymatic assays were carried out in pH 6.5 buffer.
For the measurements of K_m/V_max, screening of the protease inhibitor library, as well as IC₅₀ measurements, proteolytic reaction with 100 nM M^pro in 100 µL of pH 6.5 reaction buffer was carried out at 30 °C in a Cytation 5 imaging reader (Thermo Fisher Scientific) with filters for excitation at 360/40 nm and emission at 460/40 nm. Reactions were monitored every 90 s. For K_m/V_max measurements, a FRET substrate concentration ranging from 0 to 200 µM was applied. The initial velocity of the proteolytic activity was calculated by linear regression for the first 15 min of the kinetic progress curves. The initial velocity was plotted against the FRET concentration with the classic Michaelis–Menten equation in Prism 5 software. For the screening of protease inhibitor library and IC₅₀ measurements, 100 nM M^pro was incubated with protease inhibitor at 30 °C for 30 min in reaction buffer, then the reaction was initiated by adding 10 µM FRET substrate, the reaction was monitored for 1 h, and the initial velocity was calculated for the first 15 min by linear regression. The IC₅₀ was calculated by plotting the initial velocity against various concentrations of protease inhibitors by use of a dose-response curve in Prism 5 software. Proteolytic reaction progress curve kinetics measurements with GC376, MG132, boceprevir, calpain inhibitor II, and calpain inhibitor XII used for curve fitting, were carried out as follows: 5 nM M^pro protein was added to 20 µM FRET substrate with various concentrations of testing inhibitor in 200 µL of reaction buffer at 30 °C to initiate the proteolytic reaction. The reaction was monitored for 4 h. The progress curves were fit to a slow binding Morrison equation (Eq. (3)) as described previously^{18 ,42 (link)}: $E+I\underset{k_1}{\overset{K_2}{\leftrightarrow }}EI\underset{k_1}{\overset{K_2}{\leftrightarrow }}E{I}^{*}$ ${\mathrm{K}}_{\mathrm{I}}={\mathrm{k}}_{-1}/{\mathrm{k}}_1$ $\mathrm{P}\left(\mathrm{t}\right)={\mathrm{P}}_0+{\mathrm{V}}_{\mathrm{s}}\mathrm{t}-\left(\mathrm{Vs}-{\mathrm{V}}_0\right)\left(1-e^{-\mathrm{kt}}\right)/\mathrm{k}$ $\mathrm{k}={\mathrm{k}}_2\left[\mathrm{I}\right]/\left({\mathrm{K}}_{\mathrm{I}}+\left[\mathrm{I}\right]\right)$ where P(t) is the fluorescence signal at time t, P₀ is the background signal at time 0, V₀, V_s, and k represent, respectively, the initial velocity, the final steady-state velocity and the apparent first-order rate constant for the establishment of the equilibrium between EI and EI*.^{42 (link)} k₂/K_I is commonly used to evaluate the efficacy for covalent inhibitor. We observed substrate depletion when proteolytic reactions progress longer than 90 min; therefore only first 90 min of the progress curves were used in the curve fitting (Fig. 6, middle column). In this study, we could not accurately determine the k₂ for the protease inhibitors: calpain inhibitor II, MG132, boceprevir, and calpain inhibitor XII, due to the very slow k₂ in these cases: significant substrate depletion before the establishment of the equilibrium between EI and EI*. In these cases, K_I was determined with Morrison equation in Prism 5.