Fig. 1: The WEHI-P series is a novel scaffold for PLpro inhibitors.

a High Throughput Screening cascade for the identification of PLpro inhibitors. Schematic top, Ub-Rhodamine110 (UbRh) assay was used for assessing the biochemical inhibition (IC₅₀) of PLpro. Rhodamine110 is cleaved off the ubiquitin moiety by PLpro which releases a fluorescent signal that accumulates proportional to activity and can be measured at 535 nm. Below, a diverse library of 412,644 compounds was screened in a single concentration (29.16 µM) with one replicate. 966 compounds (hit rate of 0.23%) were identified from the primary screen. These compounds were then assessed in a 10-point titration study in the PLpro assay (confirmation) and USP21 assay (counter) in duplicate. 20.7% of primary hits, or 200 compounds, had confirmed activity in the PLpro assay. 11.5% of hits from the primary screen, or 111 compounds, showed activity in the USP21 counter-screen assay. 16 compounds displayed no activity against USP21 and were selective for PLpro. b Screening hit WEHI-P1 was optimised to WEHI-P4. The WEHI-P1 core structure represents a novel scaffold not seen in any other PLpro inhibitor and exhibits a methoxynaphthyl group bridged by a ketone (in red) to a piperidine with a substituted cyclopentyl group (in blue). Replacement of the ketone to an O-methyloxime in WEHI-P2 generated activity in the cellular FRET assay (11 µM). Replacing the cyclopentyl with a pendant cyclohexanol and enantiomer separation generated WEHI-P4 with potent biochemical, cellular and antiviral activity. Compounds were assessed for biochemical (IC₅₀, UbRh, Supplementary Fig. 2a) cellular (FRET EC₅₀, Supplementary Fig. 2c), and SARS-CoV-2 plaque assay activity (Antiviral EC₅₀, Supplementary Fig. 2d), and by SPR (K_D, Supplementary Fig. 2g). c Crystal structures of PLpro bound to WEHI-P1 and WEHI-P4 were determined in different space groups. The BL2 region of PLpro is coloured yellow. The BL2 loop is not involved in WEHI-P1 binding and participates in a crystal contact (Supplementary Fig. 3a-c, Supplementary Table 1). In WEHI-P4 the BL2 region is in the ‘closed’ conformation to encompass the compound. A zoomed-in view of PLpro bound to WEHI-P4 is shown with key residues labelled. d Structure of WEHI-P4 (top) and GRL0617 (bottom, PDB: 7JIR³⁴) with proteins under a semi-transparent surface. The WEHI-P series induces a conformational change of PLpro Met208 to expose a pocket not seen in the GRL-0617 bound structure or any other published PLpro inhibitor complex structures. Figure 1a Created in BioRender⁹².