José Gallego

In the 3′ untranslated region of betacoronaviruses, a pseudoknot involving stem–loop 3SL2 has been proposed to form a binding site for the viral RNA polymerase enabling the initiation of negative-sense RNA synthesis during the processes of virus replication and discontinuous transcription. Based on bioinformatic analyses and a mutagenesis strategy monitored with electrophoretic experiments and viral replication assays, we report that, in addition to the local pseudoknot in the 3′UTR, 3SL2 can establish a distal tertiary contact with an exposed sequence in the ORF7b gene located 1847 nucleotides upstream in the SARS-CoV-2 genome. The bases involved in this contact are highly conserved in all SARS-like coronaviruses and sequencing data indicate that this motif may form an alternative polymerase binding site enabling the synthesis of non-canonical subgenomic RNA. These findings provide a foundation for investigating the molecular mechanisms governing the processes of replication, discontinuous transcription, and recombination in these viruses.

The SARS-CoV-2 frameshift stimulation element (FSE) is a critical RNA structure that is essential for viral replication and represents a promising target for antiviral intervention. Here, Chemical Cross-Linking and Isolation by Pull-down (Chem-CLIP) covalent target validation and binding site mapping was applied, to identify small-molecule binding pockets within the FSE and ultimately develop a ligandability map. These studies employed ∼ 190 Chem-CLIP fragments, including the fluoroquinolone merafloxacin, previously shown to interact with this element. Covalent mapping defined merafloxacin's binding pocket at a nucleotide-level resolution and revealed interactions that, along with structure-based design, efficient one-pot on-plate synthesis and competitive displacement assays, enabled the development of bioactive compounds with antiviral activity. Complementary chemical probing with dimethyl sulfate (DMS) in the presence of a bioactive ligand, coupled to Deconvolution of RNA Alternative Conformations (DRACO), revealed that compound binding increased the reactivity of specific nucleotides with DMS, indicative of changes in local RNA folding. These results highlight the importance of combining Chem-CLIP and DMS profiling to differentiate direct ligand binding from ligand-induced changes in RNA structure. In addition, in silico pocket analysis of FSE structures derived from cryogenic-electron microscopy (cryo-EM) studies identified four recurring cavities, including the experimentally determined merafloxacin and Chem-CLIP fragments binding pockets. Altogether, the findings advance our understanding of RNA-ligand interactions and support a strategy to design and discover small molecules that bind RNA structures.

Protein–protein interactions (PPI) frequently involve α-helices and are challenging targets for small-molecule drugs. Here we report the design, synthesis and evaluation of new PPI inhibitors based on a bilaterally substituted p-terphenyl scaffold. The side groups of this scaffold are projected in a broad spatial angle and reproduced the interactions of the myosin A (MyoA) α-helix wrapped by the Myosin Tail Interacting Protein (MTIP) in Plasmodium parasites causing malaria. Fluorescence, calorimetry, and NMR spectroscopy analyses revealed that the terphenyl molecules recognized the MyoA binding site within the MTIP and were capable of displacing the α-helix from its protein receptor and triggering comparable conformational changes in MTIP. The MTIP affinity of the best inhibitor was strikingly close to that exhibited by the MyoA helix. These data indicate that a small-molecule terphenyl compound can efficiently mimic a four-times heavier polypeptide. These molecules may serve as probes for PPIs involving deeply buried α-helices.

The programmed ribosomal frameshift (PRF) region is found in the RNA genome of all coronaviruses and shifts the ribosome reading frame through formation of a three-stem pseudoknot structure, allowing the translation of essential viral proteins. Using NMR spectroscopy, comparative sequence analyses and functional assays we show that, in the absence of the ribosome, a 123-nucleotide sequence encompassing the PRF element of SARS-CoV-2 adopts a well-defined two-stem loop structure that is conserved in all SARS-like coronaviruses. In this conformation, the attenuator hairpin and slippery site nucleotides are exposed in the first stem-loop and two pseudoknot stems are present in the second stem-loop, separated by an 8-nucleotide bulge. Formation of the third pseudoknot stem depends on pairing between bulge nucleotides and base-paired nucleotides of the upstream stem-loop, as shown by a PRF construct where residues of the upstream stem were removed, which formed the pseudoknot structure and had increased frameshifting activity in a dual-luciferase assay. The base-pair switch driving PRF pseudoknot folding was found to be conserved in several human non-SARS coronaviruses. The collective results suggest that the frameshifting pseudoknot structure of these viruses only forms transiently in the presence of the translating ribosome. These findings clarify the frameshifting mechanism in coronaviruses and can have a beneficial impact on antiviral drug discovery.

Human immunodeficiency virus-type 1 (HIV-1) remains one of the leading contributors to the global burden of disease, and novel antiretroviral agents with alternative mechanisms are needed to cure this infection. Here, we describe an exploratory attempt to optimize the antiretroviral properties of benfluron, a cytostatic agent previously reported to exhibit strong anti-HIV activity likely based on inhibitory actions on virus transcription and Rev-mediated viral RNA export. After obtaining six analogs designed to modify the benzo[c]fluorenone system of the parent molecule, we examined their antiretroviral and toxicity properties together with their capacity to recognize the Rev Recognition Element (RRE) of the virus RNA and inhibit the RRE–Rev interaction. The results indicated that both the benzo[c] and cyclopentanone components of benfluron are required for strong RRE–Rev target engagement and antiretroviral activity and revealed the relative impact of these moieties on RRE affinity, RRE–Rev inhibition, antiviral action and cellular toxicity. These data provide insights into the biological properties of the benzo[c]fluorenone scaffold and contribute to facilitating the design of new anti-HIV agents based on the inhibition of Rev function.

Antiviral agents are needed for the treatment of SARS-CoV-2 infections and to control other coronavirus outbreaks that may occur in the future. Here we report the identification and characterization of RNA-binding compounds that inhibit SARS-CoV-2 replication. The compounds were detected by screening a small library of antiviral compounds previously shown to bind HIV-1 or HCV RNA elements with a live-virus cellular assay detecting inhibition of SARS-CoV-2 replication. These experiments allowed detection of eight compounds with promising anti-SARS-CoV-2 activity in the sub-micromolar to micromolar range and wide selectivity indexes. Examination of the mechanism of action of three selected hit compounds excluded action on the entry or egress stages of the virus replication cycle and confirmed recognition by two of the molecules of conserved RNA elements of the SARS-CoV-2 genome, including the highly conserved S2m hairpin located in the 3’-untranslated region of the virus. While further studies are needed to clarify the mechanism of action responsible for antiviral activity, these results facilitate the discovery of RNA-targeted antivirals and provide new chemical scaffolds for developing therapeutic agents against coronaviruses.