Protein-protein interactions as therapeutic target in RNA viruses.

Abstract

Chapter 1 provides an overview of the existing literature on protein-protein interactions and their role in the life cycle of plus-strand RNA viruses with focus on Alphaviruses and Coronaviruses. The first part of the chapter delves into a comprehensive overview of the Chikungunya virus (CHIKV) life cycle, genomic organization, detailed functions of structural and non-structural proteins, epidemiology, and details about reported in vitro and in vivo CHIKV inhibitors. An in-depth description of the capsid protein, its structural details, multi-faceted roles, and its involvement in virus particle maturation and budding process has been done. Furthermore, the chapter also explains the essential role of protein-protein interactions in the life cycle of alphaviruses. The second part briefly gives an overview of SARS-CoV-2, and describes the life cycle of SARS-CoV-2. The vital role of RBD-ACE2 interaction in the entry of virus particles into host cells is emphasized. The essential RBD-ACE2 interaction as an ideal target for the development of therapeutics against SARS-CoV-2 has been explained in detail with previously reported entry inhibitors against coronaviruses. Chapter 2 reports identifying and evaluating the antiviral potential of thymoquinone, a natural compound targeting Chikungunya virus (CHIKV) capsid protein. The capsid protein (CP) of CHIKV is a multifunctional protein with a conserved hydrophobic pocket that plays a crucial role in the capsid assembly and virus budding process. This study demonstrates the antiviral activity of thymoquinone, a natural compound targeting the hydrophobic pocket of CP. The binding of thymoquinone to the hydrophobic pocket of CHIKV CP was analyzed using structure based molecular docking, isothermal titration calorimetry, and fluorescence spectroscopy. The binding constant KD obtained for thymoquinone was ~27 µM. Additionally, cell-based antiviral studies showed that thymoquinone diminished CHIKV replication with a half maximal concentration (EC50) value of ~4.4 µM. Reduction in viral RNA copy number and viral replication as assessed by the quantitative reverse transcription polymerase chain reaction (qRT PCR) and immunofluorescence assay (IFA), confirmed the antiviral potential of thymoquinone. Our study reveals that thymoquinone is an effective antiviral targeting the hydrophobic pocket of CHIKV CP and may serve as the basis for the development of a broad-spectrum therapy against alphavirus diseases. I Chapter 3 describes the anti-CHIKV activity of efavirenz investigated by in vitro cell culture based antiviral assay, IFA and qRT-PCR. These studies demonstrated dose-dependent robust anti-CHIKV activity of efavirenz at low micromolar concentration (EC50 = ~1.33 µM). To determine potential broad anti-alphavirus activity of efavirenz, its inhibitory activity against the Sindbis virus was detected. Interestingly, efavirenz also inhibited the replication of SINV at a low micromolar range (EC50 = ~0.7 µM). Further, several approaches were employed to investigate the possible mechanism of anti-viral activity of efavirenz. Time of addition assay, direct transfection of virus replicon RNA and viral negative sense RNA specific RT-PCR elucidated that efavirenz inhibits the early stage of viral replication after virus entry by strongly interfering with the synthesis of negative-sense viral RNA. Chapter 4 reports the important role of RBD-ACE2 interaction in the entry of SARS-CoV-2 into the cells. Viral attachment and entry are of particular interest among possible protein-protein interaction targets for therapeutics development in the life cycle of viruses because it is first step in the replication of viruses. This chapter reports the evaluation of SARS-CoV-2 entry inhibition activity of GR 128935 hydrochloride hydrate identified by structure-assisted drug design and high-throughput virtual library screening against the protein-protein interaction of RBD-ACE2. The binding of GR 128935 hydrochloride hydrate against the ACE2 were evaluated by the Surface plasmon resonance (SPR) (KD = ~3.9 µM). The inhibition of RBD-ACE2 interaction was proved by ELISA and entry inhibiting ability of GR 128935 hydrochloride hydrate was demonstrated with the help of pseudovirus entry inhibition assay (IC50 = ~0.61 µM). Finally, GR 128935 hydrochloride hydrate inhibited the SARS-CoV-2 replication in Vero cells with the EC50 value of ~0.8 µM. Chapter 5 comprehensively concludes the findings in the thesis, representing a multi-faceted strategy in addressing the challenges posed by emerging viruses of global healthcare concern, CHIKV and SARS-CoV-2. The first study evaluated the affinity of thymoquinone against the hydrophobic pocket of CHIKV CP, followed by an assessment of anti-CHIKV potential in cell culture-based studies, opening up the possibilities for further in vivo investigations. The next study focuses on repurposing HIV antiretroviral drug efavirenz against CHIKV, as robust anti CHIKV activity of efavirenz was observed in cell culture-based antiviral assays. The mechanism by which efavirenz exerts the antiviral activity was investigated and was found that it inhibited the synthesis of minus-strand viral RNA. This supports and suggests that efavirenz modulates the early stage replication processes in the virus life cycle. The third study in thesis confirms GR 128935 hydrochloride hydrate as an entry inhibitor of SARS-CoV-2, after detailed investigation II by SPR, RBD-ACE2 ELISA, pesudovirus inhibition assay and cell culture based anti-SARS CoV-2 assay. In conclusion, the present study, for the first time, identified thymoquinone and efavirenz as inhibitors of CHIKV replication. Further, in the case of SARS-CoV-2, the entry inhibition activity of GR 128935 hydrochloride hydrate was validated using various techniques. Out of these three inhibitors thymoquinone and GR 128935 hydrochloride hydrate are potential protein-protein interaction inhibitors. These identified compounds hold potential as therapeutic treatments for infections of their respective target pathogens, CHIKV and SARS-CoV-2.