SARS-CoV-2 Nucleocapsid Protein Is a Potential Therapeutic Target for Anticoronavirus Drug Discovery

Abstract

SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, is a highly contagious positive-sense RNA virus. Its explosive community spread and the emergence of new mutant strains have created palpable anxiety even in vaccinated people. The lack of effective anticoronavirus therapeutics continues to be a major global health concern, especially due to the high evolution rate of SARS-CoV-2. The nucleocapsid protein (N protein) of SARS-CoV-2 is highly conserved and involved in diverse processes of the virus replication cycle. Despite its critical role in coronavirus replication, N protein remains an unexplored target for anticoronavirus drug discovery. Here, we demonstrate that a novel compound, K31, binds to the N protein of SARS-CoV-2 and noncompetitively inhibits its binding to the 5' terminus of the viral genomic RNA. K31 is well tolerated by SARS-CoV-2-permissive Caco2 cells. Our results show that K31 inhibited SARS-CoV-2 replication in Caco2 cells with a selective index of ~58. These observations suggest that SARS-CoV-2 N protein is a druggable target for anticoronavirus drug discovery. K31 holds promise for further development as an anticoronavirus therapeutic. IMPORTANCE The lack of potent antiviral drugs for SARS-CoV-2 is a serious global health concern, especially with the explosive spread of the COVID-19 pandemic worldwide and the constant emergence of new mutant strains with improved human-to-human transmission. Although an effective coronavirus vaccine appears promising, the lengthy vaccine development processes in general and the emergence of new mutant viral strains with a potential to evade the vaccine always remain a serious concern. The antiviral drugs targeted to the highly conserved targets of viral or host origin remain the most viable and timely approach, easily accessible to the general population, in combating any new viral illness. The majority of anticoronavirus drug development efforts have focused on spike protein, envelope protein, 3CL^pro, and M^pro. Our results show that virus-encoded N protein is a novel therapeutic target for anticoronavirus drug discovery. Due to its high conservation, the anti-N protein inhibitors will likely have broad-spectrum anticoronavirus activity.

Keywords: RNA virus; antiviral agents; coronavirus; nucleocapsid protein; virus replication.

FIG 1

K31 binds to the SARS-CoV-2 N protein and inhibits interaction with the 5′ terminus of viral genomic RNA. (A) Schematic diagram of the genome organization of SARS-CoV-2 (IVDC-HB-01/2019 HB01 strain) (1). (B) Schematic diagram of domain representation of SARS-CoV-2 N protein (33, 34). RBD, RNA binding domain. (C) Purification of C-terminally His-tagged SARS-CoV-2 N protein using Ni-NTA chromatography. (D) Binding profiles for the interaction of SARS-CoV-2 N protein with 5′ NCR sequence (open circles) and 5′ UTR of hantaviral mRNA (filled red circles) using a filter binding assay. The inset shows a double reciprocal plot. For details about the filter binding assay and generation of binding profiles, please see references , , and . a.u., arbitrary units. (E) Inhibition profiles showing the percentage of radiolabeled 5′ NCR-N protein complex retained on the filter at increasing input concentrations of K31 (open circles) and inactive compound 100605 (filled red circles), previously identified in a high-throughput screen (16, 17). (F) The data from panel E were used to calculate percent inhibition at each input concentration of K31 or compound 100605. The data points were fitted to a dose-response curve using Origin 6.0 Pro for the calculation of EC₅₀ values. (G) Biolayer interferometry showing the association and dissociation kinetics for the binding of purified N protein to the immobilized biotinylated 5′ NCR in the absence (orange) and presence (purple) of K31. (H) Binding profiles for N protein-5′ NCR interaction at four different K31 concentrations, as shown. (I) The Lineweaver-Burk plots were generated using the data from panel H. (J) The secondary plot was generated by plotting the slopes of Lineweaver-Burk plots from panel I versus input K31 concentration. The data points were fitted to a straight line.

FIG 2

K31 binds to SARS-CoV-2 N protein and inhibits virus replication in cells. (A) Biolayer interferometry showing the association and dissociation kinetics for the binding of K31 with C-terminally His-tagged N protein, immobilized on a Ni-NTA biosensor. The experiment was repeated at three different concentrations of K31 as shown. (B) Cytotoxicity of K31 on Caco2 cells. Caco2 cells in 96-well plates were treated with increasing concentrations of K31 for 3 days and examined for cytotoxicity using a CellTiter-Glo luminescent assay (see reference for details). (C) Inhibition profile showing the percentage of SARS-CoV-2 replication in Caco2 cells at increasing concentrations of K31. SARS-CoV-2 replication was determined by quantification of viral genomic RNA using real-time PCR. (D) Western blot analysis showing N protein levels in SARS-CoV-2-infected Caco2 cells in the absence or presence of 10 μM K31.