Targeting Two-Tetrad RNA G-Quadruplex in the SARS-CoV-2 RNA Genome Using Tetraphenylethene Derivatives for Antiviral Therapy.

Targeting the specific RNA conformations that are crucial for SARS-CoV-2 replication is a viable antiviral approach. The SARS-CoV-2 genome contains GG repeats capable of forming unstable two-tetrad G-quadruplex (GQ) structures, which exist as a mix of conformations, including hairpin (Hp), intra-, and intermolecular GQs. RGQ-1, originating from the nucleocapsid gene's ORF, adopts a dynamic equilibrium of conformations, including intramolecular hairpin and G-quadruplex (Hp-GQ) structures, as confirmed by CD analysis. In this study, tetraphenylethene (TPE) derivatives were developed to target the Hp-GQ conformational equilibrium of RGQ-1. EMSA, fluorescence spectroscopy, and ITC assays confirmed that two TPE derivatives, TPE-MePy and TPE-Allyl Py, bind to RGQ-1. CD thermal melting experiments indicate that RGQ-1 is stabilized by 8.56 and 12.54 °C in the presence of TPE-MePy and TPE-Allyl Py, respectively. Additionally, luciferase assays demonstrated that TPE derivatives suppressed luciferase activity by 2.2-fold and 3.6-fold, respectively, shifting the HpGQ equilibrium toward the GQ conformation, as suggested by CD spectroscopy. Treatment of SARS-CoV-2-infected A549 cells with TPE derivatives reduced the levels of viral RNA, spikes, and nucleocapsid proteins. To explore their antiviral mechanism, preinfection and postinfection treatments were tested, revealing that the TPE derivatives specifically suppressed the postentry stages of viral replication without affecting viral entry. These findings highlight the therapeutic potential of TPE derivatives in inhibiting key gene expressions critical for SARS-CoV-2 replication.