Targeting the Main Protease (M^pro, nsp5) by Growth of Fragment Scaffolds Exploiting Structure-Based Methodologies

Area of Science:

• Biochemistry
• Drug Discovery
• Structural Biology

Background:

• The SARS-CoV-2 main protease (Mpro), also known as nsp5, is a critical enzyme for viral replication and a key drug target.
• Existing antiviral strategies require new therapeutic agents to combat the ongoing pandemic and potential future outbreaks.

Purpose of the Study:

• To identify and develop novel inhibitors targeting the SARS-CoV-2 main protease (Mpro).
• To explore fragment-based drug discovery using NMR spectroscopy to find starting points for Mpro inhibitors.

Main Methods:

• Primary screening of four compound libraries using Nuclear Magnetic Resonance (NMR) spectroscopy.
• Fragment-based drug design involving synthesis and characterization of derivatives, including compound 35b.
• Co-crystallization of compound 35b with Mpro to elucidate its binding mode.

Main Results:

• Identified Z604, a uracil-containing fragment with time-dependent binding to Mpro, sensitive to reducing conditions.
• Synthesized and characterized 13 related compounds, leading to the development of the four-armed compound 35b.
• Co-crystallization revealed that 35b binds non-covalently, occupying all four active site subpockets of Mpro.

Conclusions:

• The NMR-derived fragment-to-hit pipeline is effective for identifying Mpro inhibitors.
• Compound 35b represents a promising starting point for developing novel SARS-CoV-2 Mpro inhibitors.
• The detailed structural information of 35b binding to Mpro can guide further drug optimization efforts.