Structural and Mechanistic Perspectives on SARS-CoV-2 Nonstructural Protein 14-Mediated Cap Formation and Drug Discovery

Design, structure-based optimization and antiviral evaluation of potent inhibitors for the macrodomain Mac1 of SARS-CoV-2

Enzymatically active macrodomains of (+)ss-RNA viruses mediate immune evasion by countering ADP-ribosylation and are therefore promising druggable targets. Here we report testing of ADP / ADP-ribose analogues for their ability to inhibit Mac1 of SARS-CoV-2, measurement of the affinity of active compounds and characterization of their binding mode by cocrystallization, uncovering critical molecular determinants of protein-ligand interaction. Key findings of the resulting structure-activity relationship (SAR) include that inhibitory potency is improved by either replacing the distal ribose of ADP-ribose by a small alkyl group or the adenine N7 by carbon. Based on insights from the SAR, we show β-methyl-GS-441524-diphosphate as nanomolar inhibitor that exhibits >1000-fold selectivity over human MacroD1 and MacroD2. Addition of C11-acyloxybenzyl (AB)-masking groups yields a membrane permeable, lipophilic prodrug that inhibits SARS-CoV-2 in cell culture (EC50 0.06 µM) while exhibiting low cytotoxicity (CC50 > 50 µM). Replacement of the terminal methyl phosphate with an ethyl phosphonate increases stability of the prodrug with little effect on toxicity and antiviral potency (EC50 = 0.03 µM), making it a membrane-permeable nucleotide-based prodrug against viral macrodomains. The authors report a complete structure-activity relationship of Mac1 of SARS-CoV-2, that was used to design potent nucleotide inhibitors. Hits were developed into membrane-permeable, non-toxic prodrugs, which strongly suppress viral replication.

Identification of the SARS-CoV-2 genome packaging signal in the nsp12-coding region

Selective genome packaging is a critical step for RNA viruses, which must distinguish genomic RNA from other abundant transcripts. For SARS-CoV-2, the cis-acting packaging signal is thought to be recognized by the nucleocapsid (N) protein, but its identity and mechanistic basis for selective recognition remain undefined. Here we identify the packaging signal within the nsp12 polymerase-coding region. CLIP-seq maps N-bound sites and, together with virus-like particle assays, pinpoints a conserved segment with strong packaging activity. An orthogonal defective-interfering RNA approach confirms its role in genome selection. We further delineate two critical subregions, α and β, that engage the N C-terminal domain. Synonymous mutations in either subregion selectively disrupt packaging and reduce viral fitness. Notably, the α subregion encompasses the ribosomal frameshifting element, revealing its dual role in viral translation and assembly. These findings establish the mechanistic basis for SARS-CoV-2 genome packaging and offer a potential antiviral target. Here, Park et al. identify a SARS-CoV-2 packaging signal within the nsp12 coding region and show that the nucleocapsid protein mediates selective genome packaging through its C-terminal domain. Synonymous mutations disrupt packaging and reduce viral fitness without affecting genome replication.

Identification of potential inhibitors of dengue virus ns5 methyltransferase and polymerase domains through virtual screening and molecular dynamics studies

Structural Features of a Tiny Viral Protein, ORF7b of SARS-CoV-2

Accessory proteins of SARS-CoV-2 play crucial roles in viral pathogenesis, yet their structural properties remain elusive. ORF7b, a small accessory protein comprising only 43 amino acids, is widely assumed to parallel the structure–function relationships of its SARS-CoV ortholog based solely on sequence homology. In this study, we challenge this paradigm through direct physicochemical and structural characterization. Sequence analysis and electrostatic profiling reveal that the SARS-CoV-2 protein is a macromolecular polyanion with a net charge of −4 at neutral pH, featuring a diffuse negative surface that is highly responsive to pH changes. Complete 3D structures generated via ab initio modeling display a helical core flanked by two highly fluctuating, disordered termini. Residue Interaction Network (RIN) topology and Normal Mode Analysis (NMA) identified specific hinges governing these flexible extremities. Furthermore, the calculated dipole moment vector is tilted outward by 24°, misaligning with the central axis. Molecular dynamics simulations suggest that while the soluble structure is highly stable in water, it undergoes severe distortions and insufficient solvation within a membrane-mimetic environment. Thermodynamic association profiles and verified interactomic data from BioGRID reveal a strong propensity for ORF7b to participate in liquid–liquid phase transitions alongside human and viral partners. Taken together, these unique properties suggest that ORF7b operates as a dynamic peripheral membrane protein rather than a sedentary transmembrane component, providing a fresh framework for future therapeutic targeting. Overall, these in silico findings shift the current paradigm on ORF7b2 topology and provide a robust, physically grounded framework that identifies specific molecular priorities for future in vitro and in vivo validation.

In Silico Evaluation of Potential SARS-CoV-2 RNA-Dependent RNA Polymerase (RdRp) Inhibitors Derived from Philippine Natural Products

RNA-dependent RNA-polymerase (RdRp), one of the key enzymes in the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) life cycle, is a recognized druggable target for Coronavirus disease 19 (COVID-19) treatment. Its inhibition has been associated with reduced viral loads in infected individuals. A library of 1562 Philippine Natural Compounds was screened in silico for potential SARS-CoV-2 anti-RdRp activity using a high-throughput virtual screening (VS) approach. Molecular docking experiments and in silico absorption, distribution, metabolism, and excretion (ADME) predictions were used to determine compounds with potential inhibitory capabilities against RdRp. The top three compounds, Vitelignin A (VIT), Vitexoside (VIX), and Cannabifolin C (CAN), were subjected to molecular dynamics (MD) simulations in complex with RdRp, confirming the formation of stable protein-ligand complexes. Overall, these results suggest promising inhibitory capabilities of these three compounds against SARS-CoV-2 RdRp. 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.

Drug Functional Site-Unknown Molecular Targets Exemplified by SARS-CoV-2 pseudoknot and ribosomal frameshifting-modulators

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.