Hi @EKDavies, the details of the assays are included in the latest update: but I can repost these detailed write-ups below if that helps. I can’t say that I have noticed the trend regarding molecular size in RapidFire (not that I have looked extensively). One think we have noticed, is that for compounds where RapidFire and Fluorescence generally agree on inhibition, RapidFire generally gives pIC50 values about half a unit more potent.
Fluorescence MPro assay:
Compounds were seeded into assay-ready plates (Greiner 384 low volume 784900) using an Echo 555 acoustic dispenser, and DMSO was back-filled for a uniform concentration in assay plates (maximum 1%). Screening assays were performed in duplicate at 20 µM and 50 µM. Hits of greater than 50% inhibition at 50 µM were confirmed by dose response assays. Reagents for Mpro assay reagents were dispensed into the assay plate in 10 µl volumes for a final of 20 µl. Final reaction concentrations were 20 mM HEPES pH=7.3, 1mM TCEP, 50 mM NaCl, 0.01% Tween-20, 10% glycerol, 5 nM Mpro, 375 nM fluorogenic peptide substrate ([5-FAM]-AVLQSGFR-[Lys(Dabcyl)]-K-amide). Mpro was pre-incubated for 15 minutes at room temperature with compound before addition of substrate. Protease reaction was measured continuously in a BMG Pherastar FS with a 480/520 ex/em filter set.
RapidFire MPro assay:
Inhibitor compounds are dispensed into 384-well plates using the ECHO dispenser (DMSO concentration < 1%). Enzyme solution, containing 20 nM Mpro, 20 mM HEPES, pH 7.5 and 50 mM NaCl, is added to each well and incubated with the inhibitor for 15 min at RT. The reaction is initiated with the addition of 2.0 μM substrate (TSAVLQSGFRK, custom synthesized in Schofield group). After 10 min the reaction is quenched with 10% formic acid and injected into an Agilent RapidFire LC-MS system. Data analysis are done with PRISM and CDD. All compounds are triaged by testing calculating the % inhibition at 5 and 50 μM final concentration. Dose response curves are done with 11 datapoints range of 100 – 0.0017 μM inhibitor.