The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been associated with causing the coronavirus disease 2019 (COVID-19) pandemic that resulted in extensive deaths and economic consequences (Nicola et al., 2020). Despite efforts to combat the virus through numerous mechanisms, few licensed drugs have been used to treat infections of SARS-CoV-2. (Brady et al., 2024). The most promising small molecules target either viral proteins like RNA-dependent RNA polymerase (RdRp) protein or, hydroxychloroquine (Chakraborty et al., 2021, Eslami et al., 2020), lopinavir (Cao et al., 2020), remdesivir (Malin et al., 2020), and Nirmatrelvir (Owen et al., 2021) against proteases, which have shown potential inhibitory activity against the SARS-CoV-2. Similarly, a number of drugs have been designed to target viral proteins directly in other viruses. (Aggarwal et al., 2017, Fatma et al., 2020, Sharma et al., 2018). Apart from these mentioned compounds, Suramin (Boniardi et al., 2023) and Chicoric acid (Mercaldi et al., 2022) are well proven promising antiviral compounds against the N-terminal domain (NTD) and C-terminal domain (CTD), respectively of SARS-CoV-2 nucleocapsid (N) protein. However, the available drugs are associated with limitations like toxicity towards various organs and potential incompatibility with other medicaments (Heskin et al., 2022) therefore, there is a need for the new drug candidates that are associated with more effectiveness and lesser side effects.

The SARS-CoV-2 virus has a positive-sense single-stranded RNA genome that encodes for 16 non-structural proteins (nsps), which include the proteases: Main proteases (Mpro) and papain-like proteases (PLpro) (Yan et al., 2022). The nsps are considered to play a role in viral replication, thus valuable for developing antivirals (Choudhary et al., 2022, Rani et al., 2022, Singh et al., 2023). Besides the nsps, four structural proteins include Spike (S), envelop (E), membrane (M), and the nucleocapsid (N) protein that play crucial roles in protecting the viral genome and ensuring the pathogenicity of viruses (Yan et al., 2022). Among structural proteins, N plays a crucial role in the packaging of the viral RNA genome into a helical ribonucleoprotein (RNP) complex (Cubuk et al., 2021). The N-protein of SARS-CoV-2 protein is approved antiviral drug against its obvious role in viral life cycle, including the replication, transcription, and encapsidation of the viral genome (McBride et al., 2014, Papageorgiou et al., 2016). N-protein aids in genome packing and results in the formation of the RNP complex on the formation of a link between viral RNA and N-protein (McBride et al., 2014). The viral RNA interacts directly with the N-protein through its two functional domains, NTD and CTD, interlinked by highly disordered linker regions (Gordon et al., 2020, Morse et al., 2023). The earlier reported crystal structures of NTD and CTD have given clear insight into the crucial residues involved in the RNA encapsidation process, thus regulating transcription (Sola et al., 2011) (Yang et al., 2021). The structural results on CTD of N-protein (N-CTD) have revealed an important role in the control of viral replication and transcription after specific contact with the transcriptional regulatory sequence (TRSs) of viral genome (Sola et al., 2015, Wu et al., 2014). The critical role that N-CTD plays in identifying conserved regulatory sequences of the SARS-CoV-2 genome and participating in the oligomerization process makes it a great target for inhibitors that might disrupt its oligomerization and RNA binding activity.

Recent structural studies have greatly contributed to our understanding of the mechanism of N-CTD RNA and small molecule binding (Mercaldi et al., 2022, Rafael Ciges-Tomas et al., 2022). For example, the complex of N-CTD with chicoric acid crystal structure explained the mechanism of action for this small molecule (Mercaldi et al., 2022). Considering the significance of this method, it is essential to identify novel inhibitor binding sites on the N-CTD that can interfere with its oligomerization and RNA binding activities. Earlier, three inhibitors, ceftriaxone, cefuroxime, and ampicillin, were screened as possible candidates targeting the N-protein of SARS-CoV-2 by using high-throughput chip screening technique (Hu et al., 2021).

In this work, we first characterized the binding affinities of ceftriaxone, cefuroxime, and ampicillin to N-CTD by using isothermal titration calorimetry (ITC). Then, using a fluorescence intensity-based RNA binding assay and fluorescence polarisation (FP) assay, we evaluated their effect on RNA binding activity. Additionally, we determined the crystal structures of N-CTD in complex with ceftriaxone (2.0 Å) and ampicillin (2.2 Å), along with the high-resolution apo structure (1.4 Å). These findings reveal the molecular interactions between these inhibitors and the N-CTD protein, illustrating their potential to disrupt RNA binding and interfere with the assembly of the viral genome into the RNP complex.