Structure-based virtual screening and experimental validation of the discovery of inhibitors targeted towards the human coronavirus nucleocapsid protein.

Nucleocapsid protein (NP), an essential RNA-binding viral protein in human coronavirus (CoV)-infected cells, is required for the replication and transcription of viral RNA. Recent studies suggested that human CoV NP is a valid target for antiviral drug development. Based on this aspect, structure-based virtual screening targeting nucleocapsid protein (NP) was performed to identify good chemical starting points for medicinal chemistry. The present study utilized structure-based virtual screening against human CoV-OC43 using the Zinc database, which is performed through docking with varying precisions and computational intensities to identify eight potential compounds. The chosen potential leads were further validated experimentally using biophysical means. Surface plasmon resonance (SPR) analysis indicated that one among the potential leads, 6-chloro-7-(2-morpholin-4-yl-ethylamino) quinoxaline-5,8-dione (small-compound H3), exhibited a significant decrease of RNA-binding capacity of NP by more than 20%. The loss of binding activity was manifested as a 20% decrease in the minimum on-rate accompanied with a 70% increase in the maximum off-rate. Fluorescence titration and X-ray crystallography studies indicated that H3 antagonizes the binding between HCoV-OC43 NP and RNA by interacting with the N-terminal domain of the NP. Our findings provide insight into the development of new therapeutics that disrupt the interaction between RNA and viral NP in the HCoV. The discovery of the new compound would be an impetus to design novel NP inhibitors against human CoV.

Structural basis for the identification of the N-terminal domain of coronavirus nucleocapsid protein as an antiviral target.

Coronaviruses (CoVs) cause numerous diseases, including Middle East respiratory syndrome and severe acute respiratory syndrome, generating significant health-related and economic consequences. CoVs encode the nucleocapsid (N) protein, a major structural protein that plays multiple roles in the virus replication cycle and forms a ribonucleoprotein complex with the viral RNA through the N protein's N-terminal domain (N-NTD). Using human CoV-OC43 (HCoV-OC43) as a model for CoV, we present the 3D structure of HCoV-OC43 N-NTD complexed with ribonucleoside 5'-monophosphates to identify a distinct ribonucleotide-binding pocket. By targeting this pocket, we identified and developed a new coronavirus N protein inhibitor, N-(6-oxo-5,6-dihydrophenanthridin-2-yl)(N,N-dimethylamino)acetamide hydrochloride (PJ34), using virtual screening; this inhibitor reduced the N protein's RNA-binding affinity and hindered viral replication. We also determined the crystal structure of the N-NTD-PJ34 complex. On the basis of these findings, we propose guidelines for developing new N protein-based antiviral agents that target CoVs.

Crystal structure-based exploration of the important role of Arg106 in the RNA-binding domain of human coronavirus OC43 nucleocapsid protein.

Human coronavirus OC43 (HCoV-OC43) is a causative agent of the common cold. The nucleocapsid (N) protein, which is a major structural protein of CoVs, binds to the viral RNA genome to form the virion core and results in the formation of the ribonucleoprotein (RNP) complex. We have solved the crystal structure of the N-terminal domain of HCoV-OC43 N protein (N-NTD) (residues 58 to 195) to a resolution of 2.0Å. The HCoV-OC43 N-NTD is a single domain protein composed of a five-stranded ß-sheet core and a long extended loop, similar to that observed in the structures of N-NTDs from other coronaviruses. The positively charged loop of the HCoV-OC43 N-NTD contains a structurally well-conserved positively charged residue, R106. To assess the role of R106 in RNA binding, we undertook a series of site-directed mutagenesis experiments and docking simulations to characterize the interaction between R106 and RNA. The results show that R106 plays an important role in the interaction between the N protein and RNA. In addition, we showed that, in cells transfected with plasmids that encoded the mutant (R106A) N protein and infected with virus, the level of the matrix protein gene was decreased by 7-fold compared to cells that were transfected with the wild-type N protein. This finding suggests that R106, by enhancing binding of the N protein to viral RNA plays a critical role in the viral replication. The results also indicate that the strength of N protein/RNA interactions is critical for HCoV-OC43 replication.

Oligomerization of the carboxyl terminal domain of the human coronavirus 229E nucleocapsid protein.

The coronavirus (CoV) N protein oligomerizes via its carboxyl terminus. However, the oligomerization mechanism of the C-terminal domains (CTD) of CoV N proteins remains unclear. Based on the protein disorder prediction system, a comprehensive series of HCoV-229E N protein mutants with truncated CTD was generated and systematically investigated by biophysical and biochemical analyses to clarify the role of the C-terminal tail of the HCoV-229E N protein in oligomerization. These results indicate that the last C-terminal tail plays an important role in dimer-dimer association. The C-terminal tail peptide is able to interfere with the oligomerization of the CTD of HCoV-229E N protein and performs the inhibitory effect on viral titre of HCoV-229E. This study may assist the development of anti-viral drugs against HCoV.