This 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 known functional sites.
Abstract
The –1 Programmed Ribosomal Frameshifting (–1 PRF) 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 modulators preferentially stabilize the remote stem (stem 3) of the PK, promoting variety of intermediate force species with the beginning base pairs of stem 1 being refolded, even after those base pairs have been unwound by ribosome. The refold of the tips of the stem 1 in turn “push back” the ribosome on the slippery sequence to result in enhanced frameshifting. On the other hand, Feff-suppressing modulators rigidify early stem regions, increasing resistance to ribosomal progression and increase the drop-off rate, eventually leading to a reduced –1 frame to 0 frame ratio in translation. Together, these findings provide the first integrated demonstration of how small molecules can modulate –1 PRF by altering RNA PK 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 known functional sites.
ABSTRACT Programmed −1 ribosomal frameshifting (−1 PRF) is a translational recoding mechanism used by many RNA viruses to regulate the expression of viral replication proteins. In coronaviruses, including SARS-CoV-2, −1 PRF is controlled by a conserved frameshift stimulation element containing a three-stemmed RNA pseudoknot located downstream of a slippery sequence. Studies have shown that conformational dynamics, mechanical stability, and structural variability of the pseudoknot influence ribosome pausing and frameshifting efficiency, identifying viral RNA structures as potential antiviral targets. This review outlines the structural organization, mechanistic basis, and conformational dynamics of viral frameshifting pseudoknots, with emphasis on the SARS-CoV-2 frameshift stimulation element. Advances in cryo-electron microscopy, single-molecule biophysics, molecular dynamics simulations, and computational modeling have identified multiple pseudoknot conformations involved in translational recoding and ribosome–RNA interactions. RNA-targeted therapeutic approaches used to suppress or modulate −1 PRF are also discussed, including small-molecule RNA binders, antisense oligonucleotides, peptide nucleic acids, and ribonuclease-targeting chimeras. These approaches act on distinct aspects of RNA structure, conformational flexibility, and stability to inhibit viral translation or promote selective RNA degradation. Major challenges include selective targeting of highly dynamic RNA structures, optimization of intracellular delivery, and minimizing off-target effects. Integration of structural biology, computational modeling, and RNA-targeted therapeutic strategies may support the development of next-generation antivirals targeting conserved viral RNA regulatory elements.
Neha Jeena, I. Khan· Antimicrobial Agents and Che...· 0 citations
A fragment-based drug discovery strategy combined with crystallographic screening and structural similarities among the fragments provide a robust structural framework for the rational design of PLpro inhibitors and support the development of novel antiviral therapeutics.
Tingting Wu, Zheng Zhou, Xingyu Li et al.· Journal of Biological Chemis...· 0 citations
Despite advances in vaccines and supportive therapies, a targeted treatment for COVID-19 remains lacking, prompting researchers to explore specific pharmacological interventions to fill this gap. In this quest, we targeted the main protease (Mpro) of SARS-CoV-2, a well-established druggable target crucial for viral replication and transcription. 714 antiviral compounds were computationally screened against the active-site residues of Mpro, and 17 hits showed significantly better interactions with Mpro than the selected control (N3). The two top-scoring compounds, LY2784544 and Pimobendan, were further evaluated through 100-ns molecular dynamics (MD) simulations. Comprehensive post-simulation analyses, including root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), radius of gyration, solvent-accessible surface area, principal component analysis, free energy landscape (FEL) mapping, and MM/PBSA binding free energy calculations, demonstrated stable complex formation and persistent engagement of the catalytic dyad (His41–Cys145) and S1/S2 subsites. Molecular Mechanics/Poisson–Boltzmann Surface Area (MM/PBSA) results indicated favorable binding free energies, with Pimobendan showing comparatively stronger energetic stabilization. The study identifies LY2784544 and Pimobendan as potential antiviral agents for Mpro. Given their stability, strong binding energy (BE) profiles, and existing clinical data, these compounds warrant additional experimental validation to determine their potential efficacy against SARS-CoV-2.
M. Alam, Lina I. Alnajjar, T. K. Upadhyay et al.· Journal of King Saud Univers...· 0 citations
Structural and biochemical studies have elucidated the architecture of the nsp14 N7-methyltransferase domain, revealing an S-adenosyl-L-methionine (SAM)-dependent fold with a defined cofactor-binding site and an adjacent cap-binding pocket that orients the RNA substrate for methyl transfer.
Yifan Zhao, Rhea Guo, Yang Yang et al.· Microorganisms· 0 citations
Results suggest promising inhibitory capabilities of these three compounds against SARS-CoV-2 RdRp, and their shared presence in extracts of Vitex negundo, a Philippine medicinal plant, warrants further in vitro and in vivo investigation regarding the anti-SARS-CoV-2 activity of its extracts and active components.
A. Ang, Alexandra P Lee, J. Billones et al.· COVID· 0 citations
The restoration of mutant p53 stability is a highly sought-after strategy in targeted cancer therapy. This study presents a structure-based virtual screening and molecular dynamics approach to nominate FDA-approved drugs as candidate stabilizers of mutant p53 for downstream experimental validation. A virtual screening library of FDA-approved compounds was docked against three representative p53 mutants (7DHY, 7DHZ, and 7V97) to evaluate their binding potential. The prioritized candidates demonstrated consistent, multi-conformer binding affinities. Protein–ligand interaction profiling revealed that the candidate DB09280 possesses a highly dense interaction network, particularly against the V272M and R249S variants. Residue-level analysis of the G245S structural mutant showed that DB09280 uniquely engages His19, a crucial residue for zinc coordination, and forms stabilizing contacts with adjacent flexible loop residues, including ASN35 and PRO32. Subsequent 500 ns molecular dynamics simulations were consistent with DB09280 acting as a putative conformational clamp on the timescale sampled. The ligand-bound (holo) system exhibited substantially reduced global structural drift (RMSD) and attenuated local residue fluctuation (RMSF) within the core domain compared to the highly unstable apo state. Principal component analysis further indicated that DB09280 restricts the broad conformational sampling of the mutant into a stable, dominant basin within the sampled trajectory. Together, these computational findings nominate DB09280 as a promising candidate structural stabilizer of mutant p53 worthy of experimental follow-up. We emphasize that the in silico stabilization observed here is not equivalent to functional rescue of p53 transcriptional activity; biochemical, biophysical, and cell-based assays will be required to establish whether DB09280 restores wild-type-like DNA binding or tumor-suppressor function in mutant p53 contexts.
Mena Abdelsayed, Y. Boulaamane· International Journal of Mol...· 0 citations
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