Small molecules dually targeting SARS-CoV-2 and host G-quadruplexes: binding mechanism explored by integrated experiments and computations.

BACKGROUND

The ongoing COVID-19 pandemic caused by SARS-CoV-2 continues to pose significant challenges to global public health. Small-molecule ligands targeting G-quadruplex (G4) structures have shown considerable potential in antiviral therapy. However, existing studies have largely focused on individual viruses or isolated host systems, primarily employing in vitro or in vivo approaches to examine interactions between specific ligands and particular G4 structures. There remains a need for novel intervention strategies and a deeper mechanistic understanding of G4-ligand interactions to advance rational drug design.

RESULTS

This study proposes, for the first time, a dual-targeting strategy that simultaneously engages G4 structures in both the SARS-CoV-2 viral genome and host genes relevant to viral entry (ACE2, AXL, and TMPRSS2). Four potential G4-forming sequences from these targets were experimentally confirmed to fold into stable RNA G4 structures. Using an integrated approach of spectroscopic analysis, isothermal titration calorimetry (ITC), and molecular dynamics (MD) simulations, we systematically evaluated the binding of three small-molecule ligands, berbamine (BBM), TMPyP4 (TMP), and topotecan (TPT), to the G4s. Among them, TMP demonstrated superior G4-stabilizing capability and significantly higher binding affinity compared to BBM and TPT. Energy decomposition analysis revealed that strong electrostatic interactions contributed by the positively charged pyridine groups of TMP are critical for stable binding. These results suggest that incorporating charged functional groups into ligand scaffolds can enhance G4-binding affinity.

SIGNIFICANCE: By integrating comprehensive experimental characterization and molecular dynamics simulations to explore dual-targeting G4-ligand interactions, this work establishes a systematic methodology for in vitro G4-ligand research and provides critical insights for designing new SARS-CoV-2 therapeutics. This strategy holds significant potential not only against COVID-19 but also as a proactive blueprint for addressing future emerging viral threats.