FRET-based Assay for Protease Inhibitor Screening

The FRET-based assay was performed similarly to the self-assembled monolayers for matrix-assisted desorption/ionization mass spectrometry (SAMDI-MS) assay. Assays were performed in 20-μL volume in 384-well nonbinding low-volume plates (Greiner Bio-One, Monroe, NC) at ambient temperature. 3CLpro and its mutants were preincubated with inhibitors for 30 min. Reactions were initiated by the addition of a FRET-compatible peptide substrate, dabcyl-KTSAVLQSGFRKM-E(Edans)-NH₂. Fluorescence was measured for 90 min at 2-min intervals using 340/460-excitation/emission filters on an Envision plate reader (Perkin Elmer). The IC₅₀ values were calculated by fitting the curves using a four-parameter equation in GraphPad Prism.
To calculate the dimer dissociation constant (K_d), the velocities of enzyme titration of WT and mutant 3CLpros were fitted to equations 1 and 2.
$$V_0=V_{\max }[\mathrm{S}]/(\mathrm{Km}\hspace{0.17em}+\hspace{0.17em}[\mathrm{S}])$$
$$V_{\mathit{max}}=K_{\mathit{cat}}\left[D\right]=K_{\mathit{cat}}\frac{K_d\hspace{0.17em}+\hspace{0.17em}4C_T\hspace{0.17em}-\hspace{0.17em}\sqrt{K_d{}^2\hspace{0.17em}+\hspace{0.17em}8K_d{C}_T}}{8}$$
Equation 2 was described previously for the calculation of the monomer dimer equilibrium dissociation constant (K_d) (28 (link)).