Discovery of First-in-Class PROTAC Degraders of SARS-CoV-2 Main Protease

Abstract

We have witnessed three coronavirus (CoV) outbreaks in the past two decades, including the COVID-19 pandemic caused by SARS-CoV-2. Main protease (M^Pro), a highly conserved protease among various CoVs, is essential for viral replication and pathogenesis, making it a prime target for antiviral drug development. Here, we leverage proteolysis targeting chimera (PROTAC) technology to develop a new class of small-molecule antivirals that induce the degradation of SARS-CoV-2 M^Pro. Among them, MPD2 was demonstrated to effectively reduce M^Pro protein levels in 293T cells, relying on a time-dependent, CRBN-mediated, and proteasome-driven mechanism. Furthermore, MPD2 exhibited remarkable efficacy in diminishing M^Pro protein levels in SARS-CoV-2-infected A549-ACE2 cells. MPD2 also displayed potent antiviral activity against various SARS-CoV-2 strains and exhibited enhanced potency against nirmatrelvir-resistant viruses. Overall, this proof-of-concept study highlights the potential of targeted protein degradation of M^Pro as an innovative approach for developing antivirals that could fight against drug-resistant viral variants.

Figure 1

Representative reversible covalent inhibitors MPI8 and MPI29 of SARS-CoV-2 M^Pro. (a) M^Pro-MPI8 structure (PDB: 7JQ5). (b) M^Pro-MPI29 structure (PDB: 7S6W).

Figure 2

Design of M^Pro PROTAC degraders based on the reversible covalent inhibitors MPI8 and MPI29. (a) MPD1–6 via linking a CRBN ligand at the N-terminal phenyl group at the P4 site of MPI8. (b) MPD7–10 via linking a CRBN ligand at the N-terminal tert-butyl group of MPI29.

Figure 3

M^Pro PROTAC degraders. (a) Chemical structures of M^Pro PROTAC degraders MPD1-MPD10. (b) Inhibition curves of MPD1-MPD10 on M^Pro. Triplicate experiments were performed for each compound. For all experiments, 20 nM M^Pro was incubated with an inhibitor for 30 min before 10 μM Sub3 was added. The M^Pro-catalyzed Sub3 hydrolysis rate was determined by measuring the linear increase of product fluorescence (Ex: 336 nm/Em: 490 nm) at the initial 5 min reaction time.

Figure 4

Degradation of M^Pro by MPD1-MPD3. (a) Design of M^Pro-eGFP fusion. (b) Representative flow cytometry analysis of the potency of MPD2 in degrading M^Pro in the M^Pro-eGFP 293T stable cell line. M^Pro-eGFP cells were evaluated after being treated with different concentrations of MPD2 for 48 h. The percentage of positively expressed M^Pro-eGFP fusion protein was displayed in red at the bottom of the graph. (c, e, g) The potency of MPD1 (c), MPD2 (e), and MPD1 (g) in degrading M^Pro was evaluated in the M^Pro-eGFP 293T stable cell line by immunoblots after the cells were treated with different concentrations of MPD1, MPD2, and MPD3 for 48 h. Representative immunoblots are shown, and β-actin was used as a loading control in all immunoblot analyses. (d, f, h) The graph presents the normalized protein content in the immunoblots as mean values ± s.e.m. (n = 3) in the graph.

Figure 5

PROTAC degrader MPD2 downregulates the protein levels of M^Pro in a time-dependent manner. (a) The time course of MPD2-mediated M^Pro degradation was evaluated in the M^Pro-eGFP 293T stable cell line by immunoblots after the cells were treated with 3 μM MPD2 for various time points as indicated. (b) The graph presents the normalized protein content in the immunoblots as mean values ± s.e.m. (n = 3 biologically independent experiments) in the graph.

Figure 6

MPD2 degrades M^Pro in a ligand- and CRBN-dependent manner. (a) Pretreatment with M^Pro ligand MPI8 or CRBN ligand Pomalidomide blocks the M^Pro degradation induced by MPD2. The potency of MPD2 in degrading M^Pro was evaluated in the M^Pro-eGFP 293T stable cell line by immunoblots after the cells were treated with different concentrations of MPD2 for 48 h. (b) The normalized protein content in the immunoblots (β-actin was used as a loading control) is presented as mean values ± s.e.m. (n = 3 biologically independent experiments) in the titration curved graph. (c) CRISPR knockout of CRBN blocks MPD2-induced M^Pro degradation as shown in control (CRBN-CN) and CRBN knockout (CRBN-KO) M^Pro-eGFP 293T cells. Immunoblots evaluated the potency of MPD2 in degrading M^Pro after the cells were treated with different concentrations of MPD2 for 48 h. (d) The normalized protein content in the immunoblots (β-actin was used as a loading control) is presented as mean values ± s.e.m. (n = 3 biologically independent experiments) in the titration curved graph.

Figure 7

MPD2 degrades M^Pro in a proteasome-dependent manner and proteasome inhibition blocks the M^Pro degradation by MPD2. (a, b) A representative of 3 immunoblot analyses of M^Pro in M^Pro-eGFP 293T stable cell line after they were either pretreated with the proteasome inhibitor MG132 (1 μM) or pretreated with vehicle for 1 h and then were treated with different concentrations of MPD2 for 48 h. (c) Normalized protein content in the immunoblots (β-actin was used as a loading control) in the titration curved graph.

Figure 8

Antiviral effectiveness of PROTACs against the SARS-CoV-2 delta variant. A549-ACE2 cells were inoculated with 0.01 TCID₅₀ per cell (a) or 0.1 TCID₅₀ per cell (b) SARS-CoV-2 for 1 h and then treated with antiviral. (a) Effect of PROTACs or nirmatrelvir (Nir) treatment on virus growth 48 h after inoculation. (b) Effect of PROTACs treatment on M^Pro accumulation 48 h after inoculation with SARS-CoV-2 delta variant. M^Pro in cell lysates was detected with monoclonal antibody against 34 kDa M^Pro.

Figure 9

Antiviral Activity of MPD2 against the SARS-CoV-2 variants. MPD2 impedes SARS-CoV-2 strains WA.1 (a), BA.1 (b), and XBB.1.5 (c) infection. A549-hACE2 cells were infected with WA.1 (0.1), BA.1 (0.3), or XBB.1.5 (MOI 0.3) in the presence of various concentrations of MPD2. After 48 h postinfection, supernatant viral RNAs from each treatment dose were quantified and normalized to that of dimethyl sulfoxide (DMSO) controls. (d) MPD2 inhibits the NSP5 E166A mutant. A549-hACE2 cells were infected with the recombinant live-attenuated Δ3678 mGFP or NSP5 E166A mutant and treated with MPD2 for 48 h. mGFP-positive cells from each treatment dose were quantified and normalized to those of DMSO controls. Error bars indicate the standard deviations from duplicates or triplicates.