The -1 programmed ribosomal frameshifting (-1PRF) signal of SARS-CoV-2, driven by a conserved three-stemmed RNA pseudoknot (PK), is indispensable for viral replication and represents a structurally stable yet underexplored therapeutic target. Unlike rapidly mutating viral proteins, this RNA element offers an opportunity for durable intervention but has historically been considered “undruggable.” We developed an integrative drug discovery and characterization pipeline that combines molecular docking, molecular dynamics simulations, and dual-luciferase assays to systematically identify and validate frameshifting-efficiency (Feff) modulators from FDA-approved compounds. To move beyond traditional similarity-based screening, we introduced a contact-distribution-matching method, which ranks candidate compounds by comparing their predicted RNA interaction fingerprints with those of reference modulators. This computational approach, paired with experimental validation, enabled us to expand the repertoire of Feff modulators and establish correlations between binding patterns and functional outcomes. To uncover the underlying mechanisms, we applied steered molecular dynamics simulations and single-molecule optical tweezers measurements, revealing that Feff-enhancing compounds preferentially destabilize proximal stems of the PK, while stabilizing the distal domains, to promote alternative folding intermediates, whereas Feff-suppressing compounds rigidify early stem regions, increasing resistance to ribosomal progression. These contrasting effects reshape the RNA conformational energy landscape and directly alter its mechanical resistance during ribosomal translocation. Together, these findings provide the first integrated demonstration of how small molecules can modulate -1PRF by altering RNA pseudoknot folding dynamics. More broadly, our framework establishes a generalizable strategy for rationally targeting structured RNAs with repurposed drugs and offers new opportunities to expand the druggable genome to include noncoding RNA elements and other biomolecular targets lacking conventional active sites.
Sanad et al. (Sun,) studied this question.