Discovery and structural characterization of chicoric acid as a SARS-CoV-2 nucleocapsid protein ligand and RNA binding disruptor

Abstract

The nucleocapsid (N) protein plays critical roles in coronavirus genome transcription and packaging, representing a key target for the development of novel antivirals, and for which structural information on ligand binding is scarce. We used a novel fluorescence polarization assay to identify small molecules that disrupt the binding of the N protein to a target RNA derived from the SARS-CoV-2 genome packaging signal. Several phenolic compounds, including L-chicoric acid (CA), were identified as high-affinity N-protein ligands. The binding of CA to the N protein was confirmed by isothermal titration calorimetry, 1H-STD and 15N-HSQC NMR, and by the crystal structure of CA bound to the N protein C-terminal domain (CTD), further revealing a new modulatory site in the SARS-CoV-2 N protein. Moreover, CA reduced SARS-CoV-2 replication in cell cultures. These data thus open venues for the development of new antivirals targeting the N protein, an essential and yet underexplored coronavirus target.

Design of Tracers in Fluorescence Polarization Assay for Extensive Application in Small Molecule Drug Discovery

“…Trivella's team proposed a new antiviral screening method targeting SARS-CoV-2 nucleocapsid protein (N protein) in 2022. 35 The N protein plays an essential role in the process of transcription initiation and packaging of the viral genomic RNA. Thus, it is considered as a potential target for developing SARS-CoV-2 replication inhibitors.…”

“…For interaction-based screening, involved biomolecular interactions can be exclusively classified into protein–protein interaction (PPI), protein–DNA interaction, protein–RNA interaction, and receptor–ligand interaction (Figure B). This screening way tends to be applied as a preliminary screening method , or as a final hit screening method in the overall mixed screening process according to recent research. For example, to discover a SARS-CoV-2 nucleocapsid protein ligand and RNA binding disruptor, Mercaldi’s group employed an FP-based HTS assay to achieve primary screening from ∼3200 approved drugs and bioactive molecules and got a list of 78 compounds at the first step, which is strong evidence that FP can indeed be involved in advancing drug discovery.…”

“…This screening way tends to be applied as a preliminary screening method , or as a final hit screening method in the overall mixed screening process according to recent research. For example, to discover a SARS-CoV-2 nucleocapsid protein ligand and RNA binding disruptor, Mercaldi’s group employed an FP-based HTS assay to achieve primary screening from ∼3200 approved drugs and bioactive molecules and got a list of 78 compounds at the first step, which is strong evidence that FP can indeed be involved in advancing drug discovery. Moreover, enzyme activity based FP can be used to identify disease-related enzyme activity regulators which possess small molecular sizes and druglike properties.…”

“…FP assay can utilize a well-designed ligand with a specific nucleotide sequence to generate a probe and then evaluate protein–DNA interaction, delve into protein–RNA interaction, and measure nuclease activity (Figure A). Of note, the application of RNA probe in the FP method has made rapid progress and has become a hot spot for HTS of therapeutic agents targeting RNA binding protein (RBP). , This burgeoning field will improve the understanding of the RBP function mode and thereby advance drug discovery to treat RNA-mediated diseases, such as cancer, − Alzheimer’s disease, and viral infection …”

“…75,76 This burgeoning field will improve the understanding of the RBP function mode and thereby advance drug discovery to treat RNA-mediated diseases, such as cancer, 77−80 Alzheimer's disease, 81 and viral infection. 35 As one of the indispensable ligand types, nucleic acids have some unique edges to stand out and satisfy the requirements of a competitive binding experiment. First, sequence-specific binding between RBP/DNA binding protein (DBP) and the corresponding nucleic acid determines favorable specificity and good affinity.…”