Sequential Optimization Approach Toward an Azapeptide-Based SARS-CoV-2 Main Protease Inhibitor.

Area of Science:

• Medicinal Chemistry
• Virology
• Structural Biology

Background:

• Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a global health threat.
• The SARS-CoV-2 main protease (Mpro) is crucial for viral replication and a key antiviral target.

Purpose of the Study:

• To identify and characterize a potent azapeptide-based inhibitor of the SARS-CoV-2 Mpro.
• To evaluate the antiviral activity, safety, and stability of the identified inhibitor.

Main Methods:

• Sequential identification of small-molecule peptidomimetics with varying cysteine-reactive groups.
• Optimization of a P1-P4 azapeptide scaffold combined with identified warheads.
• Biochemical assays to determine inhibitory constants (k_inac/K_i).
• Antiviral assays (EC50), cytotoxicity assessments, and plasma stability tests.
• X-ray crystallography to elucidate the molecular interaction with Mpro.

Main Results:

• A potent Mpro inactivator (compound 12), a chloracetohydrazide derivative, was identified with a k_inac/K_i of 78,900 M^-1s^-1.
• Compound 12 demonstrated significant antiviral activity (EC50 = 0.47 µM) with no observed cytotoxicity.
• X-ray crystallography revealed irreversible inhibition via thioether linkage formation between the inhibitor and the Mpro active-site cysteine.

Conclusions:

• A novel, potent, and stable azapeptide-based Mpro inhibitor was successfully developed.
• The inhibitor exhibits promising antiviral efficacy and a favorable safety profile.
• Structural insights confirm the mechanism of irreversible inhibition, supporting further development for COVID-19 treatment.