Exploring Noncovalent Protease Inhibitors for the Treatment of Severe Acute Respiratory Syndrome and Severe Acute Respiratory Syndrome-Like Coronaviruses

Abstract

Over the last 20 years, both severe acute respiratory syndrome coronavirus-1 and severe acute respiratory syndrome coronavirus-2 have transmitted from animal hosts to humans causing zoonotic outbreaks of severe disease. Both viruses originate from a group of betacoronaviruses known as subgroup 2b. The emergence of two dangerous human pathogens from this group along with previous studies illustrating the potential of other subgroup 2b members to transmit to humans has underscored the need for antiviral development against them. Coronaviruses modify the host innate immune response in part through the reversal of ubiquitination and ISGylation with their papain-like protease (PLpro). To identify unique or overarching subgroup 2b structural features or enzymatic biases, the PLpro from a subgroup 2b bat coronavirus, BtSCoV-Rf1.2004, was biochemically and structurally evaluated. This evaluation revealed that PLpros from subgroup 2b coronaviruses have narrow substrate specificity for K48 polyubiquitin and ISG15 originating from certain species. The PLpro of BtSCoV-Rf1.2004 was used as a tool alongside PLpro of CoV-1 and CoV-2 to design 30 novel noncovalent drug-like pan subgroup 2b PLpro inhibitors that included determining the effects of using previously unexplored core linkers within these compounds. Two crystal structures of BtSCoV-Rf1.2004 PLpro bound to these inhibitors aided in compound design as well as shared structural features among subgroup 2b proteases. Screening of these three subgroup 2b PLpros against this novel set of inhibitors along with cytotoxicity studies provide new directions for pan-coronavirus subgroup 2b antiviral development of PLpro inhibitors.

Keywords: COVID-19; ISG5; PLpro; coronavirus; severe acute respiratory syndrome 2; ubiquitin.

Figure 1.. PLpro interactions with series I and II naphthalene-based inhibitors.

Western naphthyl groups are highlighted in yellow, easter arene groups are highlighted in teal, and core linker groups are highlighted in pink. A. Key hydrophobic and hydrogen bonding interactions of series I inhibitor GRL0617; B. Key hydrophobic and hydrogen bonding interactions of the series II initial hit 6577871.

Figure 2. Deubiquitination and deISGylation Activity of BtSCoV-Rf1.2004 PLpro

. Different poly-Ub linkages were tested against BtSCoV-Rf1.2004 A. At 37°C 10 μM each of M1, K6, K11, K27, K29, K33, K48, and K63 linked di-Ub were incubated with 20nM BtSCoV-Rf1.2004 PLpro. Samples were taken from the reaction tube at indicated time points. B. Under similar reaction conditions 13.65μM each of K48 and K63 linked tetra-Ub was incubated with 23nM PLpro for 3 hours with samples taken at given time points. C. Activity of BtSCoV-Rf1.2004 PLpro for proISG15 from multiple species. BtSCoV-Rf1.2004 PLpro was evaluated for the cleavage of proISG15s from the following species: human (Homo sapiens; AAH09507.1), cow (Bos taurus; NP_776791.1), vesper bat (Myotis davidii; ELK23605.1), Egyptian fruit bat (Rousettus aegyptiacus; XP_015999857.1), pig (Sus scrofa; ACB87600.1), hedgehog (Erinaceus europaeus; XP_007525810.2), mouse (Mus musculus; AAB02697.1), dromedary camel (Camelus dromedarius; XP_010997700.1), sheep (Ovis aries; AF152103.1), northern tree shrew (Tupaia belangeri; AFH66859.1), greater horseshoe bat (Rhinolophus ferrumequinum; XP_032969719.1), Chinese rufous horseshoe bat (Rhinolophus sinicus; XP_019567580.1), rabbit (Oryctolagus cuniculus; XP_017195918), and jackknife fish (Oplegnathus fasciatus; BAJ16365.1). At 37°C, 10μM of each ISG15 was incubated with 20nM of SARS-CoV-2 PLpro for at least 1hr with samples taken at the time points indicated. The summary of the proISG15 cleavage assays for BtSCoV-Rf1.2004 PLpro is presented as a heat map. Colors range from dark red (no cleavage) to green (relatively robust cleavage)

Figure 3.. Tertiary structure of subgroup 2b PLpros compared to groups 2a and 2c.

A. Cartoon representation of BtSCoV-Rf1.2004 PLpro (Green) secondary structure with helix and sheet labels corresponding to Figure S1 dssp calculations. B. Overlaid cartoon representations of BtSCoV-Rf1.2004 PLpro (Green), SARS-CoV-1 PLpro (PDB 3E9S) (Pink), and SARS-CoV-2 PLpro (PDB 7JIR) (Blue). The four PLpro domains are labeled and color coded: Fingers (Purple), Palm (Orange), Thumb (Yellow), Ubl (Cyan). C. Overlaid cartoon representations of BtSCoV-Rf1.2004 PLpro (Green), MERS-CoV PLpro (PDB 5W8T)(Yellow), and MHV PLP (5WFI)(Grey) with their Ubl domains represented by ribbons. The Red Circle denotes the Finger domain

Figure 4.. Inhibitor binding pocket of three subgroup 2b viruses.

A. A F_o-F_c electron density composite omit map is shown contoured at 3σ (green mesh). With GRL0617 shown in purple and BtSCoV-Rf1 PLpro shown in green. B. GRL0617 (purple) bound to BtSCoV-Rf1 PLpro (green cartoon) overlaid with SARS-CoV-2 (white surface and cartoon) showing a possible path to active site for future inhibitors. C. Stereoview overlay of GRL0617 bound to three different different SARS-CoV PLpros: BtSCoV-Rf1 (green and purple) (PDB 7SKQ), SARS-CoV-1 (pink and orange) (PDB 3E9S), and SARS-CoV-2 (blue and yellow) (PDB 7JIR).

Figure 5.. Crystal contacts affecting PLpro conformation (PDB 7SKQ)

A. Overlaid cartoon representations of BtSCoV-Rf1.2004 PLpro chain A (green) and chain B (blue). B. Zinc finger loops of the two domains differ due to a crystal contact being made by chain B. C. GRL0617 of chain B is shifted in the binding pocket due to a crystal contact with chain B V226.

Figure 6.. BtSCoV-Rf1 PLpro in complex with 37 (PDB 7SKR)

A. An F_o-F_c electron density composite omit map is shown contoured at 3σ (green mesh). With 37 shown in blue and BtSCoV-Rf1 PLpro shown in raspberry. B. Overlay of 1 (teal) bound to SARS-CoV-2 PLpro (yellow).

Figure 7.. Cytotoxicity Studies of Inhibitors

A. CC₅₀ values of PLpro inhibitors in human cell lines after 48 hrs. B. Testing of Compound 37 for Cytotoxicity Vero E6 cells were plated at 6 × 10⁵ cells/well and incubated overnight at 37°C. Subsequently, the cells were washed 1x with PBS. Compound 37 drug dilutions were prepared to 100 uM, 50 uM, 25 uM, 12 uM, 6 uM, 3 uM and 1 uM in overlay media (DMEM supplemented with 1% serum). Culture media was decanted, and 1 mL of virus diluted in infection media to a MOI=0.1 was added to the cells and incubated for 1 hour at 37°C. Following incubation, the inoculum was removed, and 3mL/well of Compound 37 dilutions were added and incubated for 4 days at 37°C and 5% CO2. Control wells included a virus only, uninfected, or DMSO well. Following the 4-day incubation, cells were fixed with methanol: acetone (80:20) for 20 min at room temperature then stained with 0.2% crystal violet. Plaques were counted and analyzed by Prism8 by GraphPad.