tetano
Editor, Senior Moderator
Eur J Med Chem
. 2026 Apr 30:314:118919.
doi: 10.1016/j.ejmech.2026.118919. Online ahead of print.
Novobiocin defines an Asn28 allosteric pocket that governs SARS-CoV-2 main protease activity
Mohit Bhardwaj[SUP] 1 [/SUP], Raushan Anjum[SUP] 2 [/SUP], Sheetal Thakur[SUP] 1 [/SUP], Pradeep Sharma[SUP] 3 [/SUP], Ashok Kumar Patel[SUP] 4 [/SUP]
Affiliations
The repeated outbreaks of coronavirus show how hard it is to keep antiviral effectiveness when the virus mutates. Coronaviruses depend on tightly regulated proteolytic processing mediated by the 3-chymotrypsin-like protease (3CL[SUP]pro[/SUP]), making it a key antiviral target. Most current inhibitors engage the catalytic site, an approach that remains vulnerable to resistance driven by active-site mutations. Here, we investigate an alternative strategy based on allosteric regulation of 3CL[SUP]pro[/SUP] by targeting a pocket surrounding residue Asn28, previously shown to influence enzymatic activity and dimer stability. Structure-based virtual screening identified novobiocin as a candidate ligand for this region, which lies adjacent to but distinct from the catalytic center. Biophysical experiments showed direct binding of novobiocin to 3CL[SUP]pro[/SUP] in solution, with sub-micromolar affinity (K[SUB]d[/SUB] ∼ 3 × 10[SUP]-7[/SUP] M). Protease thermal stability and dimeric assembly were lowered by ligand binding. Enzymatic assays revealed a pronounced reduction in catalytic turnover with minimal effects on substrate binding, consistent with an allosteric mechanism of inhibition, and yielded IC[SUB]50[/SUB] values of ∼0.5 μM across independent assays. Molecular docking and simulation analyses supported stable binding at the Asn28-associated pocket and revealed localized changes in conformational dynamics. These findings show that novobiocin allosterically inhibits 3CL[SUP]pro[/SUP] and identify the Asn28-associated pocket as a relevant target for developing inhibitors with improved resistance to viral evolution.
Keywords: 3CL(pro); Allosteric modulation; Drug resistance; Kinetics; Robustness; Thermodynamics; Virtual screening.
. 2026 Apr 30:314:118919.
doi: 10.1016/j.ejmech.2026.118919. Online ahead of print.
Novobiocin defines an Asn28 allosteric pocket that governs SARS-CoV-2 main protease activity
Mohit Bhardwaj[SUP] 1 [/SUP], Raushan Anjum[SUP] 2 [/SUP], Sheetal Thakur[SUP] 1 [/SUP], Pradeep Sharma[SUP] 3 [/SUP], Ashok Kumar Patel[SUP] 4 [/SUP]
Affiliations
- PMID: 42091005
- DOI: 10.1016/j.ejmech.2026.118919
The repeated outbreaks of coronavirus show how hard it is to keep antiviral effectiveness when the virus mutates. Coronaviruses depend on tightly regulated proteolytic processing mediated by the 3-chymotrypsin-like protease (3CL[SUP]pro[/SUP]), making it a key antiviral target. Most current inhibitors engage the catalytic site, an approach that remains vulnerable to resistance driven by active-site mutations. Here, we investigate an alternative strategy based on allosteric regulation of 3CL[SUP]pro[/SUP] by targeting a pocket surrounding residue Asn28, previously shown to influence enzymatic activity and dimer stability. Structure-based virtual screening identified novobiocin as a candidate ligand for this region, which lies adjacent to but distinct from the catalytic center. Biophysical experiments showed direct binding of novobiocin to 3CL[SUP]pro[/SUP] in solution, with sub-micromolar affinity (K[SUB]d[/SUB] ∼ 3 × 10[SUP]-7[/SUP] M). Protease thermal stability and dimeric assembly were lowered by ligand binding. Enzymatic assays revealed a pronounced reduction in catalytic turnover with minimal effects on substrate binding, consistent with an allosteric mechanism of inhibition, and yielded IC[SUB]50[/SUB] values of ∼0.5 μM across independent assays. Molecular docking and simulation analyses supported stable binding at the Asn28-associated pocket and revealed localized changes in conformational dynamics. These findings show that novobiocin allosterically inhibits 3CL[SUP]pro[/SUP] and identify the Asn28-associated pocket as a relevant target for developing inhibitors with improved resistance to viral evolution.
Keywords: 3CL(pro); Allosteric modulation; Drug resistance; Kinetics; Robustness; Thermodynamics; Virtual screening.