Molecular mechanism of a specific capsid binder resistance caused by mutations outside the binding pocket

• Single-site mutations cause high-resistance (HR) to capsid-binding inhibitors.
• Two new HR amino acid substitutions were identified in viral capsid protein 1.
• I1207K and I1207R are located outside the ligand binding site.
• Both substitutions push the neighboring R1095 into the hydrophobic pocket.
• This ultimately leads to the abolishment of drug action.

Enteroviruses cause various acute and chronic diseases. The most promising therapeutics for these infections are capsid-binding molecules. These can act against a broad spectrum of enteroviruses, but emerging resistant virus variants threaten their efficacy. All known enterovirus variants with high-level resistance toward capsid-binding molecules have mutations of residues directly involved in the formation of the hydrophobic binding site. This is a first report of substitutions outside the binding pocket causing this type of drug resistance: I1207K and I1207R of the viral capsid protein 1 of coxsackievirus B3. Both substitutions completely abolish the antiviral activity of pleconaril (a capsid-binding molecule) but do not affect viral replication rates in vitro. Molecular dynamics simulations indicate that the resistance mechanism is mediated by a conformational rearrangement of R1095, which is a neighboring residue of 1207 located at the heel of the binding pocket. These insights provide a basis for the design of resistance-breaking inhibitors.