Figure 3.

(A) Dalcetrapib directly inhibits SARS-CoV-2 3CLpro activity. The IC₅₀ for dalcetrapib was determined by measuring fluorescence released from the quenched peptide exposed to the protease. Dalcetrapib was first de-esterified by basic hydrolysis of the ester bond to obtain dalcetrapib-thiol, which was then preincubated with 3CLpro for 24 h before a further 30 min preincubation in the presence of 1 mM DTT. Slopes of the relative fluorescence unit (RFU) increase were derived after the first 15 min of incubation with the peptide. Curve-fitting was performed using a GraphPad Prism four-parameter method. (B) Procedure for the assessment of the reversibility of 3CLpro inhibition by dalcetrapib. 3CLpro was incubated for 24 h with a buffer (control) or dalcetrapib-thiol produced as indicated in the method section. The incubation mixtures were then diluted or not 1000-fold through three ultrafiltration cycles to remove free dalcetrapib-thiol and concentrate 3CLpro. Samples labeled #1, #2, #3, and #4 were analyzed for 3CLpro activity (shown in panel C). (C) Assessment of the reversibility of 3CLpro inhibition by dalcetrapib. Protease activity “before” and “after” repeated dilution/filtration cycles. Dalcetrapib-thiol was preincubated with 3CLpro for 24 h at room temperature in the presence of 1 mM DTT before addition of the quenched peptide substrate. Slopes of the relative fluorescence unit (RFU) increase were derived after the first 15 min of incubation with the peptide.