SARS-CoV-2 M^pro inhibitors and activity-based probes for patient-sample imaging

Area of Science:

• Biochemistry
• Virology
• Drug Discovery

Background:

• The emergence of SARS-CoV-2 has led to the COVID-19 pandemic, with no effective antiviral treatments currently available.
• The SARS-CoV-2 main protease (Mpro) is a critical enzyme for viral replication and a promising target for antiviral drug development.

Purpose of the Study:

• To identify substrate preferences of SARS-CoV and SARS-CoV-2 main proteases.
• To design and synthesize potent inhibitors and activity-based probes for SARS-CoV-2 Mpro.
• To provide a structural basis for developing novel antiviral agents and diagnostic tools.

Main Methods:

• Synthesis of a combinatorial library of fluorogenic substrates with glutamine at the P1 position.
• Enzyme kinetics to determine substrate preferences of SARS-CoV and SARS-CoV-2 Mpro.
• Design, synthesis, and structural analysis (crystal structure) of SARS-CoV-2 Mpro inhibitors and activity-based probes.
• Visualization of active SARS-CoV-2 Mpro in infected patient cells.

Main Results:

• Characterization of substrate preferences for SARS-CoV and SARS-CoV-2 Mpro.
• Development of a potent SARS-CoV-2 inhibitor with a half-maximal effective concentration (EC50) of 3.7 µM.
• Creation of two activity-based probes, including one with a determined crystal structure complexed with SARS-CoV-2 Mpro.
• Successful visualization of active SARS-CoV-2 Mpro within nasopharyngeal epithelial cells of COVID-19 patients.

Conclusions:

• The study provides a structural framework for designing effective SARS-CoV-2 inhibitors.
• Developed inhibitors and probes can serve as a basis for new antiviral therapies and diagnostic tests for COVID-19.
• Understanding SARS-CoV-2 Mpro is key to combating the ongoing viral pandemic.