Structure-Based Design of Covalent SARS-CoV-2 Papain-like Protease Inhibitors.

Area of Science:

• Virology
• Drug Discovery
• Biochemistry

Background:

• Severe Acute Respiratory Syndrome-Coronavirus 2 (SARS-CoV-2) is an evolving RNA betacoronavirus responsible for the COVID-19 pandemic.
• SARS-CoV-2 encodes a papain-like protease (PLpro), a critical enzyme for viral replication and pathogenesis.
• PLpro also targets host proteins, including ubiquitin and ISG15, influencing the host immune response.

Purpose of the Study:

• To design and synthesize novel covalent inhibitors targeting the SARS-CoV-2 PLpro.
• To evaluate the enzymatic inhibition and antiviral activity of the designed compounds.
• To assess the in vivo pharmacokinetic properties and structural basis of inhibition.

Main Methods:

• Structure-based drug design leveraging the Val70Ub binding site of PLpro.
• Synthesis of covalent inhibitors by linking cysteine-reactive warheads to biarylphenyl scaffolds.
• Enzymatic inhibition assays (IC50) and cell-based antiviral assays (EC50).
• In vivo pharmacokinetic studies and X-ray crystallography.

Main Results:

• Several designed covalent PLpro inhibitors demonstrated potent enzymatic inhibition (IC50 = 0.1–0.3 μM) and antiviral activity (EC50 = 0.09–0.96 μM).
• Fumaramide inhibitors Jun13567, Jun13728, and Jun13714 exhibited favorable in vivo pharmacokinetic profiles.
• X-ray crystallography confirmed covalent linkage between the inhibitor Jun13567 and the catalytic Cys111 residue of PLpro.

Conclusions:

• The developed covalent PLpro inhibitors represent a promising new class of antiviral drug candidates against SARS-CoV-2.
• The design strategy targeting the Val70Ub binding site is validated by structural and functional data.
• These inhibitors offer a potential therapeutic avenue to combat the ongoing COVID-19 pandemic and future coronavirus threats.