Fig. 1: Discovery process of tricyclic PLpro inhibitors.
a Chemical structures of Hit-1 (1a), 2a, 2b, 2c 2 d and design strategy of tricyclic PLpro inhibitors by rational fragment merging. IC50 values were determined using a FRET-based enzymatic assay measuring viral polyprotein cleavage inhibition, and ΔTm values were obtained from differential scanning fluorimetry (DSF). b 1a (blue) partially overlaps with the benzamide group of classical PLpro inhibitor GRL0617 (pink) through molecular superposition. c Dose-activity curves of 2a, 2b, 2c, 2 d and GRL0617 against SARS-CoV-2 PLpro in the FRET assay. d Differential scanning fluorimetry analysis of the effect of 2a, 2b, 2c, 2 d and GRL0617 on SARS-CoV-2 PLpro stability. Exposure of hydrophobic residues monitored by an increase in relative fluorescence units (RFUs). Curves represent the average of three experiments. e Structural optimization regions of compound 2b and the molecular structure and drug properties of YL1004. IC50 values were determined by FRET enzymatic assay. Cytotoxicity CC50 was determined in Vero E6 cells with 72-hour incubation. Data in a, c, d, and e are presented as mean ± SD (n = 3 biological replicates), with the exception of the GRL0617 treated group in d (n = 2). Images in (b) were processed by using PyMOL (https:// pymol.org).
