STUDIES OF POTENTIAL SARS-CoV-2 ANTIVIRALS AGAINST NUCLEOTIDE-BINDING POCKETS OF VIRUS-SPECIFIC PROTEINS

Abstract

Chapter 1 reviews the literature related to SARS-CoV-2. This chapter provides a concise overview of the betacoronavirus genus, the SARS-CoV-2 life cycle, transmission, viral genome structure, and the function of all structural and non-structural proteins. In-depth descriptions of the RdRp protein, the function of several motifs, the structure of various channels, and their polymerization activity have been given. This chapter also covers antiviral research on SARS-CoV-2 RdRp proteins. Chapter 2 reports the nucleotide-binding pockets (NBPs) in virus-specific proteins for several viral diseases. Here, the multi-targeting attempt to identify effective antivirals has been made against NBPs in nsp12, nsp13, nsp14, nsp15, nsp16, and nucleocapsid (N) proteins of SARS-CoV-2. A structure-based drug repurposing in silico screening approach with ADME analysis identified small molecules targeting NBPs in SARS-CoV-2 proteins. Further, isothermal titration calorimetry experiments validated the binding of top-hit compounds to the purified N-protein. Cell-based antiviral assays revealed antiviral potency for INCB28060, darglitazone, and columbianadin with EC50 values of 15.71 μM, 5.36 μM, and 22.52 μM, respectively. These effective antivirals targeting multiple proteins are envisioned to direct the development of antiviral therapy against SARS-CoV-2 and its emerging variants. Chapter 3 describes the high-throughput assay development against the RdRp protein complex of SARS-CoV-2. This study is validated by an RNA gel-based assay for SARS-CoV-2 RdRp activity. High throughput assay (HTS) is critical for screening antiviral molecules; thus, the procedures must be quick, inexpensive, and robust, with excellent reproducibility. The nucleic acid stain was used for this assay, the most sensitive fluorescent stain for detecting nucleic acids. This approach can potentially be sensitive and economical to the high-throughput platform for finding RdRp inhibitors and turning them into antiviral compounds that selectively target the polymerase protein. Furthermore, we tested the selected inhibitors against SARS-CoV-2 and validated them using a cell culture-based assay. Chapter 4 reports the in-silico screening of inhibitors targeting the RdRp complex of SARS-CoV-2. The screening method has frequently been demonstrated to be effective in meeting the unique problems of antiviral drug discovery. The PyRx tool screens different virtual compound libraries to cut down on the number of drug molecules to a smaller group of potential candidates, which are then tested experimentally. In this study, the selected promising compounds were tested against SARS-CoV-2 in-vitro for antiviral drug discovery. Chapter 5 describes the in vitro antiviral studies of halogenated flavonoid synthesized compounds against the NBPs of nsP3 macrodomain of the chikungunya virus, a negative sense ssRNA virus. nsP3 has a natural ligand ADP-ribose that binds to its macrodomain, which is highly conserved. The docking of these compounds into the nsP3 macrodomain indicated that the halogenated dihydrorugosaflavonoids interacted and fitted more effectively in NBPs. The ability of halogenated dihydrorugosaflavonoids to bind to nsP3 invigorated us to evaluate these compounds for antiviral activity, which was confirmed using the cytopathic effect assay and plaque reduction assay. The results of the quantitative RT-PCR assay supported the hypothesis that 5c and 5d can reduce the viral RNA level in cells following viral infection. Furthermore, the results of an immunofluorescence assay were used to confirm the inhibitory potency of these compounds. The completed work will be significant for future research on the effects of investigated dihydrorugosaflavonoids on the RNA nucleotide-binding pockets, leading to safe and effective treatment to ward off viral infection.Chapter 6 concludes the studies in the thesis, where computer-aided structure-based virtual screening of +ssRNA virus proteins was studied using different drug libraries. Biophysical studies have shown to interact the SARS-CoV-2 protein with selected molecules. Further, the in vitro antiviral experiments have confirmed the antiviral potential of the selected molecules against the NBPs of +ssRNA viruses. We also developed the high-throughput assay against the RdRp protein complex to identify potential drug molecules.