Viral Capsid Protein - A Therapeutic Antiviral Target

Abstract

Chapter 1 is an introductory chapter, wherein we discuss the importance of targeting the viral capsid protein encasing its genetic material, consisting of repeating subunits of proteins known as protomers. The capsid performs several critical roles in protecting and delivering the viral genome and interacts with the host. The nucleocapsid (N) protein of SARS-CoV-2 is involved in forming a ribonucleoprotein (RNP) complex with the viral RNA; this is a key component of the virus life cycle for replication and transmission. With the present vaccines being not effective enough, alternative antiviral treatments are required to target the N-protein. The research aims to find inhibitors of the N-terminal (NTD) and C-terminal domains (CTD) of the N-protein. By structure-based drug repurposing, a number of compounds such as Telmisartan and BMS-189453 were found to disrupt RNA binding at the NTD with high antiviral activity. Other compounds like ceftriaxone, cefuroxime, and ampicillin were found to bind the CTD and thus inhibit RNA packaging. Further studies looked into the interaction of NTD with the host protein G3BP1 and led to new antiviral targets. The new study gives hope to new therapeutic development for SARS-CoV-2. Chapter 2 explores about the antiviral treatments for SARS-CoV-2 through targeting the RNA-binding site in the N-terminal domain (NTD) of its nucleocapsid (N) protein, which is crucial for the encapsulation of the viral genome. A structure-based drug repurposing approach identified twelve high-affinity compounds from FDA-approved drugs, natural products, and LOPAC1280 libraries. These compounds showed strong RNA-binding inhibition with IC50 values ranging from 8.8 μM to 15.7 μM. Two compounds, Telmisartan and BMS-189453, showed promising antiviral activity with EC50 values at 1.02 μM and 0.98 μM, respectively in cell-based assays. This research opened up the possibility of new therapeutic approaches against SARS-CoV-2. Chapter 3 discusses the role of SARS-CoV-2 nucleocapsid (N) protein in the packaging and replication of the viral genome. This study describe the importance of targeting the C-terminal domain, N-CTD, of the N protein, where three inhibitors were studied for the binding efficiency using isothermal titration calorimetry: ceftriaxone, cefuroxime, and ampicillin. It was observed that the best affinity was with ceftriaxone, followed by ampicillin and then cefuroxime. II Thereafter, the results of RNA-binding inhibition assays were confirmed, as their inhibitory IC50 values were between 10.4 μM and 12.4 μM. Crystal structures of N-CTD in complex with ceftriaxone and ampicillin revealed key sites residues for the interaction, further indicating the promising therapeutic target nature of N-CTD against coronaviruses, highlighting the potential of N-CTD as a therapeutic target for coronavirus treatments. Chapter 4 focuses on targeting the Ras GTPase-activating protein G3BP1 as antiviral target as G3BP1 plays crucial role in antiviral defence by forming stress granules (SGs) during viral infections. Many viruses, including SARS-CoV-2, disassemble SGs by targeting G3BP1, which helps in immune evasion. A key interaction occurs between G3BP1's NTF2-like domain and the NTD of the viral N protein. A newly identified peptide stretch (N128-138) of the N protein also directly interacts with the host G3BP1 protein. Results from this study characterised the small molecules like WIN-62577 and fluspirilene, specifically targeting the peptide-binding pocket of G3BP1. The identified molecules disrupted this protein-protein interactions (PPIs) and showed potent antiviral activity against SARS-CoV-2. These findings underscore the potential of targeting SGs proteins like G3BP1 for broad-spectrum antiviral therapies. Chapter 5 concludes this research by discussing alternative antiviral strategies against SARS-CoV-2, focusing on the N-terminal (NTD) and C-terminal (CTD) domains of the nucleocapsid (N) protein. Inhibitors targeting the RNA-binding functions of the N protein, such as Telmisartan and BMS-189453, demonstrated strong antiviral activity by disrupting RNA binding at the NTD. The CTD was targeted by ceftriaxone, cefuroxime, and ampicillin, which inhibited RNA packaging. Moreover, interfering with the interaction of NTD with host protein G3BP1 with compounds such as WIN-62577, and Fluspirilene, revealed new therapeutic avenues. These findings present promising candidates for drug repurposing and the development of novel therapies against SARS-CoV-2.