| Assay Method Information | |
| | PLpro Inhibition Assay |
| Description: | The assays were performed in 40 μL total volume in black half area 96-well plates (Greiner PN 675076) at 25° C. The assay buffer contained 20 mM Tris-HCl pH 7.45, 0.1 mg/mL bovine serum albumin fraction V, and 2 mM reduced glutathione. The final DMSO concentration in all assays was 2.5% v/v. PLpro initial rates were measured using a previously established fluorogenic peptide substrate assay (e.g., K. Ratia et al., Proc. Natl. Acad. Sci. USA, 105(42), 16119-24, 2008). The substrates Z-LRGG-AMC and Z-RLRGG-AMC were dissolved to 10 mM in DMSO and stored in aliquots at −20° C. To determine Michaelis-Menten parameters, 20 μL enzyme solution was dispensed into wells (250 nM final concentration), and reactions were initiated by adding 20 μL substrate to 0-500 μM final concentration, in triplicate. Release of aminomethylcoumarin (AMC) was monitored by a fluorescence plate reader every 50 s with an excitation wavelength of 345 nm and an emission wavelength of 445 nm, 6.25 mm read height, and gain=60. After background subtraction of the average of no-enzyme negative controls, product formation was quantified using a 0.02-5 μM calibration curve of AMC. Initial rates were determined for time points in the initial linear range by linear regression in Excel, and GraphPad Prism 9 was used to perform nonlinear regression of the Michaelis-Menten equation to the initial rate vs. substrate concentration data to yield KM and Vmax.Inhibitors were characterized by dispensing 10 μL enzyme solution into wells (115 nM final concentration), followed by 10 μL inhibitor solution at 4× desired final concentrations in 5% v/v DMSO in at least duplicate, centrifuging briefly, and incubating for 30 min. Reactions were initiated by adding 20 μL substrate to 100 μM final concentration. Initial rates were determined as described above and % residual activities were determined by normalizing to the average of no inhibitor controls (100% activity). |
| Affinity data for this assay | |
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