Novobiocin defines an Asn28 allosteric pocket that governs SARS-CoV-2 main protease activity

Area of Science:

• Virology
• Biochemistry
• Drug Discovery

Background:

• Coronaviruses mutate rapidly, challenging the effectiveness of antiviral therapies.
• The 3-chymotrypsin-like protease (3CLpro) is crucial for coronavirus replication and a primary antiviral target.
• Current 3CLpro inhibitors targeting the active site are susceptible to drug resistance mutations.

Purpose of the Study:

• To explore an alternative antiviral strategy by targeting an allosteric site on 3CLpro.
• To investigate the potential of the Asn28-associated pocket as an allosteric regulatory site.
• To evaluate novobiocin as a potential allosteric inhibitor of 3CLpro.

Main Methods:

• Structure-based virtual screening to identify potential ligands for the Asn28 pocket.
• Biophysical assays (e.g., thermal shift, binding affinity measurements) to confirm novobiocin binding.
• Enzymatic assays and molecular simulations to elucidate the inhibition mechanism and binding mode.

Main Results:

• Novobiocin directly binds to 3CLpro at the Asn28-associated pocket with sub-micromolar affinity.
• Novobiocin binding reduces protease thermal stability and dimer stability, consistent with allosteric modulation.
• Enzymatic assays demonstrate a catalytic turnover reduction (IC50 ~0.5 μM) with minimal impact on substrate binding, indicating allosteric inhibition.

Conclusions:

• Novobiocin acts as an allosteric inhibitor of 3CLpro, offering a new therapeutic avenue.
• The Asn28-associated pocket is a viable target for developing next-generation antivirals.
• This allosteric approach may yield inhibitors with enhanced resistance to viral evolution.