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Activity profiling and crystal structures of inhibitor-bound SARS-CoV-2 papain-like protease: A framework for anti–COVID-19 drug design

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Science Advances  16 Oct 2020:
Vol. 6, no. 42, eabd4596
DOI: 10.1126/sciadv.abd4596
  • Fig. 1 Activity profiling of SARS-CoV-1 and 2 PLpro protease.

    (A) Top: Schematic representation of Orf1a from SARS-hCoV-2, indicating the processed nonstructural proteins (nsps). PLpro cleavage sites are indicated by scissors. Bottom; Sequence of alignment of P4-P1’ amino acids of the indicated PLpro cleavage sites. (B) SARS-CoV-1 PLpro and SARS-CoV 2 PLpro substrate specificity profiles at the P3 and P4 positions presented as heat maps. Asterisk indicates P3 and P4 groups used throughout the study. (C) kcat/KM for tetrapeptide fluorogenic substrates toward SARS-CoV-1 PLpro and SARS-CoV-2 PLpro. (D) The rate of tetrapeptide substrate hydrolysis by DUBs. ([S] = 10 μM; SARS-CoV PLpro concentration, 0.2 μM; SARS-CoV-2 PLpro concentrations, 0.1 μM; MERS-CoV PLpro concentration, 2.5 μM; UCH-L3 concentration, 8.8 μM). (E) Tetrapeptide substrate structures. RFU/s, relative fluorescence unit per second.

  • Fig. 2 Characterization of nonnatural amino acid-containing inhibitors VIR250 and VIR251 and their crystal structures in complex with SARS-CoV-2 PLpro.

    (A and B) DUB inhibition by designed inhibitors ([I] = 2.3–300 μM; SARS-CoV PLpro concentration, 0.3 μM; SARS-CoV-2 PLpro concentration, 0.1 μM; MERS-CoV PLpro concentration, 2.5 μM; UCH-L3 concentration, 8 μM). (C) HeLa lysate selectivity assay. HeLa cell lysate first incubated for 30 min with the indicated inhibitor and next for 30 min with Biotin-Ub-VME followed by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot using streptavidin Alexa Fluor 647. (D) Top right: Schematic of the cross-linking of VIR250 and VIR251 to SARS-CoV-2 PLpro. Left: SARS-CoV-2 PLpro is shown as green ribbon representation and VIR250 is shown as spheres with carbon (orange) nitrogen (blue) oxygen (red) and sulfur (yellow). Bottom right: 2FoFc electron density map (1.0 σ) for VIR250 is shown as gray mesh. Carbon atoms of catalytic Cys111 of SARS-CoV-2 PLpro are shown as green sticks. The thioether linkage between Cys111 and VIR250 is indicated with an arrow. (E) SARS-CoV-2 PLpro/VIR251 structure presented as in (E) except with SARS-CoV-2 PLpro and VIR251 carbon atoms colored cyan and magenta, respectively. The electron density map is contoured at 1.5 σ.

  • Fig. 3 Molecular recognition of VIR250 and VIR251 by SARS-CoV-2 PLpro.

    (A) Top: Network of contacts between SARS-CoV-2 PLpro (green) and VIR250 (orange) with involved residues shown as sticks with red oxygen atoms, blue nitrogen atoms, and yellow sulfur atoms. Hydrogen bonds are indicated by dashed lines. Bottom: The VIR250 binding pocket of CoV-2 PLpro is shown as green surface with VIR250 shown as sticks. The P2-P4 positions are labeled. (B) Network of contacts between SARS-CoV-2 PLpro (cyan) and VIR251 (magenta) shown as in (A). (C) Superimposition of the CoV-2 PLpro/VIR250 and CoV-2 PLpro/VIR251 structures presented as in (A) and (B). Only the surface of Cov-2 PLpro from the PLpro/VIR251 structure is shown in the bottom panel. (D) Structure-based sequence alignment CoV-1 and CoV-2 PLpro with secondary structure was shown above sequence. Conserved residues, dots; catalytic cysteine, red star. Residues buried at the CoV-2 PLpro/VIR250 and CoV-2 PLpro/VIR251 interfaces are indicated by orange and magenta triangles below the alignment, respectively. Residues involved in contacts with the S1 Ub residue 73 to 76 (P1-P4), S1 Ub, and S2 Ub based on the SARS-CoV-2 PLpro/K48 diUb structure (PDB: 5E6J) are shaded yellow and boxed purple and cyan, respectively.

  • Fig. 4 Processing of Ub and Ubl variants by SARS-CoV-1 PLpro and SARS-CoV-2 PLpro.

    (A) SARS-CoV-1 PLpro and SARS-CoV-2 PLpro labeling by Biotin-Ub-VME. Recombinant enzymes were incubated with the indicated B-Ub-VME concentrations for 45 min at 37°C, analyzed by SDS-PAGE, and subjected to Western blot using fluorescent streptavidin Alexa Fluor 647 conjugate. (B) SARS-CoV-2 PLpro was pretreated with dimethyl sulfoxide (DMSO) or VIR251 for 30 min at 37°C and subsequently incubated with the indicated activity based Ub/Ubl probe at RT for 2 min. This was followed by SDS-PAGE and sypro staining. (C) Kinetic parameters of selected substrates for SARS-CoV PLpro and SARS-CoV-2 PLpro. Asterisk indicates that CoV-1 PLpro/ISG15-AMC pair were not analyzed in this study but kcat/KM values have previously been determined (13, 15). (D) The indicated tetraUb chains were incubated with CoV-2 PLpro for 30 min at 37°C, subjected to SDS-PAGE and sypro staining. (E) Ub-Vinyl Sulfone labeling. Ub-VS was incubated for 30 min at 37°C with the indicated protease, subjected to SDS-PAGE and sypro staining. (F) The indicated tetraUb chains were incubated with the indicated protease and analyzed as in (D).

  • Fig. 5 A molecular basis for the observed Ub/Ubl processing profiles of SARS-CoV-1 PLpro and SARS-CoV-2 PLpro.

    (A) The previously determined CoV-1 PLpro/K48 diUb complex structure (PDB: 5E6J) was superimposed onto our CoV-2 PLpro/VIR250 structure. Selected Cα atoms are shown as spheres to highlight residues involved in the different interfaces in the structures. The Cα atoms of residues contacting the S1 Ub and S2 Ub in the CoV-1 PLpro/K48 diUb complex structure are shown as purple and cyan spheres, respectively. The Cα atoms of conserved residues contacting both VIR250 and the Ub C terminus (LRGG) in the CoV-2 PLpro/VIR250 and CoV-1 PLpro/K48 diUb structures are shown as green spheres. Residues involved specifically in contacts to VIR250 and the Ub C terminus are shown as orange and yellow spheres, respectively. Magnified view of the indicated interfaces are shown to the right and residues that are different between CoV-1 and CoV-2 PLpro are written with respect to CoV-1 sequence.

Supplementary Materials

  • Supplementary Materials

    Activity profiling and crystal structures of inhibitor-bound SARS-CoV-2 papain-like protease: A framework for anti–COVID-19 drug design

    Wioletta Rut, Zongyang Lv, Mikolaj Zmudzinski, Stephanie Patchett, Digant Nayak, Scott J. Snipas, Farid El Oualid, Tony T. Huang*, Miklos Bekes, Marcin Drag, Shaun K. Olsen

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