Mechanism and inhibition of the papain‐like protease, PLpro, of SARS‐CoV‐2

A

Structure of SARS2 PLpro bound to the ubiquitin C‐terminal tail in the active site, compare with Fig 5A.

B

Superposition of ubiquitin tail in SARS2 PLpro, and compound 3j in SARS PLpro (pdb 4ovz (Baez‐Santos et al, 2014)) shows an identical binding for compounds in SARS2 PLpro and highlights the change in Tyr268/269 in SARS2 PLpro and SARS PLpro, respectively.

C, D

Compounds rac3j and rac3k, racemic versions of 3j and 3k from (Baez‐Santos et al, 2014), and their in vitro biochemical IC₅₀ values determined by the HTS assay technical triplicate and in three independent experiments (as for rac5c in Fig 5C).

E

Immunoblot characterisation of the PLpro antibody on HEK 293T cells overexpressing PLpro from MERS, SARS or SARS2. Cell lysates were immunoblotted 48 h post‐transfection. PLpro antibody is cross‐reactive with SARS and SARS2, but not MERS PLpro.

F

Immunoblot analysis showing the effect of rac5c (10 μM for 24 h) on Lys48‐polyubiquitin chain disassembly by nsp3, 48 h post‐transfection in HEK 293T cells. In this experiment, nsp3 expression was inferred by release of free GFP. Importantly, rac5c has no effect on global Lys48 chains in untransfected HEK293T cells.

G

Experiment as in Fig 5D, with a clearer effect of nsp3 on K48‐linked polyubiquitin.

Figure EV5. SARS PLpro compounds inhibit SARS2 PLpro.