VIRAL CYSTEINE PROTEASES OF +ssRNA VIRUSES AS ANTIVIRAL DRUG TARGETS.

Abstract

Chapter 1 provides an overview of the existing literature for +ss RNA viruses: Coronaviruses and Alphaviruses. The chapter provides a comprehensive overview of the SARS-CoV-2 life cycle, emerging variants, genomic organization, and detailed functions of structural and non-structural proteins. In depth description of the SARS-CoV2 cysteine protease, PLpro, its structural details, multi-faceted roles, and mechanism of proteolytic processing have been described. Furthermore, the chapter also delves into the genomic organization of alphaviruses, their life cycle dynamics, the specific functions attributed to structural and non-structural proteins, and offers an extensive literature review of the alphavirus cysteine protease nsP2 of Chikungunya virus (CHIKV). Chapter 2 reports the assessment of the inhibitory activity of natural triterpenoids: Azadirachtin, Withanolide_A, and Isoginkgetin against papain-like protease (PLpro) of SARS-CoV-2. The coronavirus disease 2019 (COVID-19) pandemic, may be over, but its variants continue to emerge, and patients with mild symptoms having long COVID are still under the radar and being investigated. SARS-CoV-2 infection leading to elevated cytokine levels and suppressed immune responses set off cytokine storm, fatal systemic inflammation, tissue damage, and multi-organ failure. Thus, drug molecules against virus-specific proteins that play a role in viral inflammation and simultaneously act on the host pathways participating in viral inflammation, is expected to provide an effective antiviral therapy against emerging SARS-CoV-2 variants of concern. Evolutionarily conserved PLpro plays an indispensable role in the virus life cycle and immune evasion. Direct-acting antivirals targeting this viral protease represent an attractive antiviral strategy that is also expected to reduce viral inflammation. The present study has evaluated the antiviral activity of natural triterpenoids: Azadirachtin, Withanolide_A, and Isoginkgetin. These molecules inhibit the PLpro proteolytic activity with half-maximal inhibitory concentrations (IC50) values ranging from ~1.42 to ~15.56 μM. Isothermal titration calorimetry (ITC) analysis validated the binding of these compounds to PLpro. As expected, the two compounds, Withanolide_A and Azadirachtin exhibit potent antiviral activity with half-maximum effective concentration (EC50) values of ~21.73 μM and ~31.19 μM, respectively. The results of this study clearly highlight the role of triterpenoids as effective antiviral molecules that target SARS-CoV-2 specific enzymes and also host immune pathways involved in virus mediated inflammation Chapter 3 reports the identification and evaluation of antivirals targeting the proteolytic activity and deubiquitinating role of PLpro. PLpro is an attractive target due to its sequence conservation and critical role in the replication and pathogenesis of SARS-CoV-2. PLpro also plays very important role in modulation of host immune responses by deubiquitinating (DUBs) or deISGylating host proteins. Thus, targeting PLpro serves as a two-pronged approach to abate SARS-CoV-2. Due to its structural and functional similarities with the host DUB enzymes, an in-house library of DUB inhibitors was constituted in this study. Five promising compounds exhibiting high binding affinities with the substrate binding site and S2-binding pocket of PLpro were identified from a library of 81 compounds with in silico screening, docking, and simulation studies. Interestingly, Lithocholic acid, Linagliptin, Teneligliptin, and Flupenthixol significantly inhibited the proteolytic activity of PLpro. Each of these compounds abrogated in vitro replication of SARS-CoV-2 with EC50 values in low micromolar range, when tested in Vero cells, HEK293T-ACE2, and A549-ACE2 cells. In addition, the crystallization and structure determination of catalytically inactive mutant of SARS-CoV-2 PLpro (C111S) in complex with Linagliptin and Lithocholic acid revealed their interactions with two distinct natural substrate binding sites of PLpro for ISG15 and ubiquitin proteins, interrupting protein-protein interactions. The findings of this study provide the proof-of-principle that the DUB inhibitors hold high potential as a new class of therapeutics against SARS-CoV-2. Additionally, this is the first study that has opened a new avenue towards not only targeting PLpro active site but also simultaneously directed towards restoration of antiviral immune response of the host for deterring SARS-CoV-2. Chapter 4 defines the signaling mechanisms by which coronaviral PLpro negatively regulates the antiviral defences of the host. To confirm whether PLpro inhibits RNA-induced IFN signaling, PLpro-transfected HKE293T cells were treated with a viral RNA mimic, poly(I:C), and the results revealed a significant reduction in IFN signaling activation in presence of PLpro. Next, our investigation delved into the functions of triterpenoid-based compounds and in house DUB inhibitors identified in preceding chapters, against the virus-triggered inflammatory pathways. The findings in this chapter unveiled the anti-inflammatory potential of identified compounds, along with their capacity to restore the diminished type-I interferon response. By revealing the additional functions of identified PLpro inhibitors, the findings shed light on the dual targeting antiviral capabilities of these compounds against SARS-CoV-2, with significant potential for further advancement in in vivo settings to fully assess their efficacy Chapter 5 focuses on identifying antiviral lead molecules by combining structure-assisted drug design and high-throughput virtual library screening, targeting the active site of nsP2 protease of Chikungunya virus (CHIKV). The global prevalence of zoonotic mosquito-borne viruses, such as CHIKV, pose increasing health threats and causes significant levels of morbidity and mortality in humans and domesticated animals. The complex spectrum of clinical and neurological manifestations of CHIKV includes acute and chronic musculoskeletal disease accompanied by high fever, rash, debilitating arthritis sometimes progressing to potentially fatal encephalitis. The lack of approved therapeutics against CHIKV is a major concern and structure-based drug designing and repurposing is an important approach that can aid in efficiently managing the infection. A prime target for drug discovery against CHIKV is nsP2 that plays a pivotal role in mediating viral assembly by cleaving polyproteins and downregulating viral restriction factors. Guided by the computational studies, the list of in silico identified and designed compounds were further shortlisted through fluorescence resonance energy transfer (FRET)-based nsP2 protease assay and the kinetic parameters were determined. Additionally, the binding of chosen compounds with the purified nsP2 protease was confirmed using ITC. Final selected compounds (LQM330, 333, and DAGII) exhibited promising broad-spectrum anti-alphavirus activity against CHIKV and Sindbis virus (SINV) using cell-based assays, strongly inhibiting the virus induced cytopathic effects and impeding the replication of the alphaviruses. Together these findings underscore the substantial development potential of the identified compounds as promising viable alternatives for the discovery of broad-spectrum anti-alphaviral therapies. Chapter 6 comprehensively concludes the findings in the thesis, representing a multi-faceted strategy in addressing the challenges posed by emerging viruses of global healthcare concern, SARS-CoV-2 and CHIKV. The first study evaluated and affirmed the inhibitory potency of two natural bioactive compounds, Azadirachtin and Withanolide_A, against proteolytic activity of PLpro followed by assessment of anti-SARS-CoV-2 potential in cell-based studies, opening up the possibilities for further structure-determination and in vivo investigations. The next set of studies focused on modes of action and structural resemblances of SARS-CoV-2 PLpro and host deubiquitinating enzymes (DUBs), resulting in development of an in house library of available DUB inhibitors. After detailed FRET, Surface Plasmon Resonance (SPR), and virus-based antiviral investigations, Linagliptin and Lithocholic acid were shortlisted as two most potent compounds. Simultaneously, the crystal structures of SARS-CoV-2 PLpro in complex with Lithocholic acid and Linagliptin were determined that provided essential insights into the structural aspects and interactions with PLpro at the atomic level. Notably, it was discovered that Linagliptin and Lithocholic acid occupied the ISG15/Ub S2-binding site of PLpro, near the thumb domain, consequently creating sterical hindrance and blocking the N-terminal domain of these substrates (ISG15 and Ub) from accessing the S2 site. Furthermore, the subsequent study evaluated the role of PLpro inhibitors on inflammatory pathways of the host. The final study identified broad-spectrum anti-alphaviral inhibitors utilizing a combined integrated computational, biophysical, biochemical, and in vitro virus based approach.