The SARS-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds

Abstract

Over 10 years have passed since the deadly human coronavirus that causes severe acute respiratory syndrome (SARS-CoV) emerged from the Guangdong Province of China. Despite the fact that the SARS-CoV pandemic infected over 8500 individuals, claimed over 800 lives and cost billions of dollars in economic loss worldwide, there still are no clinically approved antiviral drugs, vaccines or monoclonal antibody therapies to treat SARS-CoV infections. The recent emergence of the deadly human coronavirus that causes Middle East respiratory syndrome (MERS-CoV) is a sobering reminder that new and deadly coronaviruses can emerge at any time with the potential to become pandemics. Therefore, the continued development of therapeutic and prophylactic countermeasures to potentially deadly coronaviruses is warranted. The coronaviral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro), are attractive antiviral drug targets because they are essential for coronaviral replication. Although the primary function of PLpro and 3CLpro are to process the viral polyprotein in a coordinated manner, PLpro has the additional function of stripping ubiquitin and ISG15 from host-cell proteins to aid coronaviruses in their evasion of the host innate immune responses. Therefore, targeting PLpro with antiviral drugs may have an advantage in not only inhibiting viral replication but also inhibiting the dysregulation of signaling cascades in infected cells that may lead to cell death in surrounding, uninfected cells. This review provides an up-to-date discussion on the SARS-CoV papain-like protease including a brief overview of the SARS-CoV genome and replication followed by a more in-depth discussion on the structure and catalytic mechanism of SARS-CoV PLpro, the multiple cellular functions of SARS-CoV PLpro, the inhibition of SARS-CoV PLpro by small molecule inhibitors, and the prospect of inhibiting papain-like protease from other coronaviruses. This paper forms part of a series of invited articles in Antiviral Research on "From SARS to MERS: 10years of research on highly pathogenic human coronaviruses."

Keywords: 3C-like protease; MERS-CoV; Nsp3; Papain-like protease; SARS-CoV; Ubiquitin.

Fig. 1

Genome and proteome organization of SARS-CoV non-structural proteins highlighting nsp3 domain organization and PLpro cleavage sites. The ∼30 kb genome of SARS-CoV and its associated replicase, structural and accessory proteins are indicated and the sizes of boxes representing each protein are to scale (top of figure). The replicase genes encoded by ORF1a and ORF1b are shaded in grey. The nsp3 multi-domain protein is shown with the amino acids defining the approximate boundaries of each domain indicated underneath. Downstream of the SARS-CoV PLpro cleavage site between nsp2/3 (818/819aa) is a ubiquitin-like domain (Ubl-1), PDB: 2GRI, a N-terminal Glu-rich acidic-domain (AC), ADP-ribose-1″-phosphatase (ADRP) domain (PDB: 2FAV) the SARS unique domain SUD (PDB: 2WCT, 2JZE, 2KAF,), a papain-like protease (PLpro) containing a second UBl-2 domain at the N-terminus (PDB: 2FE8) followed by the nucleic acid binding domain (NAB), PDB: 2K87, the marker domain G2M and four predicted transmembrane domains (TM1–TM4) forming an additional domain containing a metal-binding region (ZF). Finally, the remainder of nsp3 is composed of so-called Y domains (Y1–3), which precede the C-terminal PLpro cleavage sequence at nsp3/4 (2740/2741aa). An alignment of the SARS-CoV PLpro cleavage sequences (right bottom corner) shows a comparison of the P-sites and P′-sites from the nsps to the C-terminal sequences of the cellular proteins ubiquitin (Ub) and ISG15 shown with an isopeptide bond at the P′-sites.

Fig. 2

Multi-domain architecture of PLpro and its two ubiquitin binding subsites. (a) The SARS-CoV PLpro monomer (PDB: 2FE8) consists of four domains: starting from N- to the C-terminus, the extended UBL, the thumb, the palm and the fingers domain. (b) An overlay of SARS-CoV PLpro (blue) with USP7 (yellow, PDB: 4M5W) displaying the USP fold. (c) A solvent-accessible surface representation of SARS-CoV PLpro is shown in blue. A K48-linked di-Ub molecule (orange) superimposed onto an ISG15 (yellow) molecule is shown with molecules represented as ribbons. The ISG15 structure consists of two tandem UBL folds (Ub1 and Ub2). The two Ub and UBL binding subsites of SARS-CoV PLpro (Ratia et al., 2014) are shown in the solvent accessible surface representation with SUb1 shaded white and SUb2 shaded yellow.

Fig. 3

Currently proposed sites of action for SARS-CoV PLpro-mediated antagonism of the innate immune response. Viral infection is sensed by RIG-I (RIG-I-like helicase) and MDA-5 (melanoma differentiation-associated protein 5) recognition of viral RNA. Recruitment of adaptor proteins MAVS transduces signals to the downstream kinase complex, which activates the transcription factor, IRF-3 and NF-κB, which coordinates the expression of type I interferons (IFN-β and -α). Type I IFN induces the activation of STAT transcription factors resulting in the expression of ISGs (IFN-stimulated genes) and the establishment of an antiviral state in surrounding cells. PLpro can act on different branches of these pathways by interacting with or recognizing and deISGylating and/or deubiquitinating proteins within these pathways. The net effect of these different functions is to help SARS-CoV evade the host antiviral response via antagonizing the establishment of an antiviral state.

Fig. 4

Catalytic center of the active site of SARS-CoV PLpro. Unliganded X-ray crystal structure of SARS-CoV PLpro (PDB: 2FE8). The catalytic triad residues, Cys112, His273 and Asp287 are shown along with the oxyanion hole-stabilizing residue Trp107 (right panel). The flexible BL2 loop that interacts with the substrates is indicated as are the zinc ion (purple sphere) and α-helix α4 in the thumb domain. Distances between atoms are given in units of Ångströms (Å) and are indicated by dashed lines.

Fig. 5

Catalytic cycle and proposed chemical mechanism of the SARS-CoV PLpro catalyzed reaction. Active site residues of the catalytic triad (Cys112, His273, Asp287) and oxyanion hole residue Trp107 are shown in black. The peptide substrate is shown in green and a catalytic water molecule is shown in blue.

Fig. 6

Examples of cysteine protease inhibitors with covalent “warheads.” The chemical “warhead” groups are shown in boxes and the reactive, electrophilic portions are colored in red, with the corresponding electrophilic “warhead” carbon indicated with an asterisk. The resulting covalent inhibitor–cysteine complexes are shown outside the boxes and the cysteine portions of the covalent adducts are colored in blue.

Fig. 7

X-ray structure of SARS-CoV PLpro in complex with ubiquitin-aldehyde (Ubal). (a) Pictorial representation of the tetrahedral hemithioaminal intermediate generated upon PLpro–Ubal complex formation, where the Ubal C-terminal residues are shown in orange and the SARS-CoV PLpro residues are shown in black. (b) X-ray crystal structure of PLpro–Ubal complex (PDB: 4MM3 (Ratia et al., 2014)) where the PLpro is shown in blue and the residues His273, Cys112, Asp287, and Trp107 are shown in ball-and-stick and colored according to atom type, and Ubal is shown in orange and colored according to atom type. Distances between atoms are given in units of ångströms (Å) and are indicated by dashed lines.

Fig. 8

SARS-CoV PLpro inhibitors identified through small-scale screening efforts. Positions within each molecule that are susceptible to covalent modification through nucleophilic attack (“warhead” positions) are indicated in red where the electrophilic carbons are indicated with an asterisk. (a) Inhibitors identified via yeast-based screen (Frieman et al., 2011), (b) thiopurine inhibitors (Chen et al., 2009, Chou et al., 2008), (c) natural product inhibitors (Cho et al., 2013, Park et al., 2012a, Park et al., 2012b), where the most potent natural product of each class is shown and the IC₅₀ detailed.

Fig. 9

Discovery and design pipeline for SARS-CoV PLpro inhibitors 24 and 15g. Results of the primary high-throughput screen identified hit compounds 7724772 (a1) and 6577871 (a2). SAR diagrams for analogs of 7724772 (b1) and 6577871 (b2) (Ghosh et al., 2010, Ratia et al., 2008). Structures of potent, first-generation leads 24 (c1) and 15g (c2). X-ray crystal structures of 24 (d1) (PDB: 3E9S) and 15g (d2) (PDB: 3MJ5) bound to SARS-CoV PLpro highlighting the residues involved in binding each inhibitor. Hydrogen bonds are shown as dashed lines.

Fig. 10

Discovery and design pipeline of compounds 3k, 3j, 3e, and 5c. (a) Initial SARS-CoV PLpro inhibitor leads, 15g and 24, (b) SARs derived from small 15g analog library, (c) top SARS-CoV PLpro inhibitor candidates, 3k, 3j, 3e, and 5c (Baez-Santos et al., 2014a).

Fig. 11

X-ray crystal structures of SARS-CoV PLpro in complex with inhibitors 3k and 3j and comparison to 15g. (a) An overlay of SARS-CoV PLpro (blue cartoon, gray surface) in complex with 3k (orange stick, PDB: 4OW0) and PLpro (cyan cartoon, gray surface) in complex with 3j (pink sticks, PDB: 4OVZ) depicting amino acids important for inhibitor binding (Baez-Santos et al., 2014a). (b) An overlay of PLpro–3k (PLpro in blue cartoon, 3k in orange sticks), PLpro–3j (PLpro in cyan cartoon, 3j in pink sticks) and PLpro–15g (green cartoon and sticks, (PDB: 3MJ5, (Ghosh et al., 2010)) complexes.

Fig. 12

X-ray crystal structures of SARS-CoV PLpro in different asymmetric units. PLpro is shown as a ribbon representation with each monomer within an asymmetric unit colored in a different shade of blue. The BL2 loop is highlighted in red and the zinc ion is shown as a pink sphere. (a) The crystallographic trimer of unliganded PLpro (PDB: 2FE8, (Ratia et al., 2006)) is shown in the left panel and a structural superposition of the three monomers is shown in the right panel. (b) The crystallographic dimer of the SARS-CoV PLpro–15g inhibitor complex (PDB: 3MJ5, (Ghosh et al., 2010)) is shown in the left panel and a structural superposition of the two monomers is shown in the right panel. Compound 15g is shown in orange. (c) The crystallographic monomer of the SARS-CoV PLpro–24 inhibitor complex (PDB: 3E9S, (Ratia et al., 2008)) is shown in the left panel. Compound 24 is shown in green. The right panel depicts the structural superposition of the SARS-CoV PLpro–15g and SARS-CoV PLpro–24 inhibitor complexes. (D) The crystallographic dimer of the SARS-CoV PLpro–3k inhibitor complex (PDB: 4OW0, (Baez-Santos et al., 2014a)) is shown in the left panel and a structural superposition of the two monomers in the asymmetric unit is shown in the right panel.

Fig. 13

X-ray crystal structures of SARS-CoV PLpro in complex with ubiquitin. (a) An overlay of the crystallographic monomer from the two PLpro–Ub complex (PDB: 4M0W, (Chou et al., 2014) and 4MM3 (Ratia et al., 2014)) crystal structures. The PLpro–Ubal complex is shown in blue with Ubal in orange and the PLproC112S–Ub complex is shown in light blue with Ub in yellow. (b) The conformational difference of the BL2 loop and the different orientations of PLpro BL2 residues and the C-terminal residues (72–76) of Ub (italics labels). (c) The different orientations of the active site residues with the distances between the atoms indicated in angstrom (black for PLpro–Ubal and red for PLproC112S–Ub).

Fig. 14

Structural comparisons of the active sites of SARS-CoV PLpro, HCoV-NL63 PLP2 and MERS-CoV PLpro. The X-ray structure of SARS-CoV PLpro in complex with compound 3k (orange ball and sticks, PDB: 4OW0) is shown superimposed with homology models of MERS-CoV PLpro and HCoV-NL63 PLP2 (green). The amino-acid residues important for SARS-CoV PLpro–inhibitor interactions are shown (blue front) along with the predicted corresponding amino acid in HCoV-NL63 PLP2 (green font) and MERS-CoV PLpro (black font). Highlighted in bold are the non-conserved substitutions in MERS-CoV PLpro sequence. Shown at the bottom of the figure is a comparison between SARS-CoV, HCoV-NL63 and MERS-CoV PLpro’s amino acid composition of the BL2 β-turn/loop (highlighted with an arrow). This loop is essential for the inhibitor induced fit mechanism of association of compound 3k and related inhibitors with SARS-CoV PLpro. This figure has been adapted from Baez-Santos et al. (2014b).