IDENTIFICATION AND CHARACTERISATION OF NOVEL CHIKV ANTIVIRAL MOLECULES TARGETING THE HOST PROTEIN G3BP1

Abstract

Alphaviruses are the members of family Togaviridae and are transmitted through the bite of an infected mosquito. Chikungunya virus (CHIKV) is a member of the Togaviridae family transmitted by Aedes albopictus and Aedes aegypti. The symptoms of CHIKV include fever, rashes, acute and chronic arthralgia. Several attempts have been made towards the development of drugs targeting the viral proteins. However, the process is costly, time-consuming and drugs often show narrow spectrum activities. Therefore, targeting the host factors using either novel compounds or repurposing drug approach seems a promising antiviral strategy against CHIKV. CHIKV has positive-sense, single-stranded, RNA genome which comprises of structural and non-structural proteins (nsPs): nsP1, nsP2, nsP3 and nsP4. Recent studies have shown the role of nsP3 in viral pathogenesis by highlighting its interaction with the host proteins such as GTPase Activating Protein SH3 Domain Binding Protein 1 (G3BP1). Molecular interaction of CHIKV nsP3 with G3BP1 host protein is important for efficient replication of CHIKV. CHIKV nsP3 protein binds to the nuclear transport factor 2 (NTF2)-like domain of G3BP1 via two tandem FGDF motifs and disrupts the stress granules (SGs) formation. Interestingly, this makes the G3BP1 host protein an additional drug target for antiviral research. In this study, seven potential compounds targeting FGDF motif binding pocket of G3BP1 have been identified using a structure-based virtual screening approach. The binding energies and binding modes of the compounds were further analyzed in detail through molecular docking and simulation studies. Surface Plasmon Resonance (SPR) and Isothermal Titration Calorimetry (ITC) experiments confirmed the binding of identified compounds to purified G3BP1 with binding affinities in micromolar (μM) range. The antiviral efficacy of compounds targeting G3BP1 was evaluated by in vitro cell culture-based antiviral studies. All seven compounds L-798106 (L-7), WIN_62577 (WIN), SB_222200 (SB2), Naltrindole hydrochloride (NAL), Dihydroergotamine (DHD), GSK1838705A (GSK), and Fluspiriline (FLU) effectively diminished the CHIKV replication with EC50 values of 1.996, 0.403, 5.387, 1.528, 7.394, 3.664, and 0.618 μM, respectively. Moreover, the CHIKV-infected cells treated with these compounds seemed to have fewer virus-induced SGs than virus-infected control cells. Interestingly, the inhibitors showed no adverse effect on SG formation in oxidative stress conditions. It further substantiated that these inhibitors effectively bind to the NTF2-like domain of G3BP1, obstruct the nsP3-G3BP1 interactions, and halt virus replication. This is the first report of compounds targeting G3BP1, the host protein playing a crucial role in CHIKV viral replication and the formation of SGs for host antiviral response. Chapter 1 reviews the literature on alphaviruses. It discusses the genome organization, the virion structure, and the life cycle of alphaviruses in detail. The formation of alphavirus polyprotein and its processing into structural and non-structural proteins has been explained. It specifically discusses the domains and conserved motifs of CHIKV nsP3 involved in host protein interactions. The significance of designing drugs and vaccine development to combat alphaviruses has been emphasized. Various cellular pathways exploited by the alphaviruses are discussed, majorly SG pathway. Various host factors manipulated by the alphaviruses for their efficient replication and spread have been described. In this study, the primary emphasis is on the host protein G3BP1 and its interaction with the FGDF motif of CHIKV nsP3. Targeting this viral-host protein interaction can lead to the development of novel antiviral strategies. Chapter 2 reports in silico approaches used to screen and identify inhibitor molecules that target the interaction of CHIKV nsP3-G3BP1 NTF2-like domain. The top seven hits have been identified based on higher binding energy using molecular docking. Further, molecular dynamic simulation studies are performed to assess the molecular dynamic parameters of ligand stability. Chapter 3 reports the expression, production and co-crystallization of the NTF2-like domain of G3BP1 host protein using a prokaryotic expression system. The protein is successfully purified using Ni2+-NTA affinity chromatography. Native protein has been crystallized. Co- crystallization of the ligands with G3BP1 NTF2-like domain has been attempted. Data collection and refinement of the protein-ligand complex have been done. However, no ligand density has been observed. Chapter 4 deals with the biophysical measurements of the proposed compounds and their binding affinities with the purified protein have been determined using ITC and SPR. Various thermodynamic parameters, such as the association constant (Ka), dissociation constant (Kd), and affinity constant (KD), are calculated using these techniques. Both techniques confirmed the strong binding affinity of the compounds to G3BP1 protein. Chapter 5 describes the in vitro evaluation of the proposed inhibitors in cell culture-based antiviral assays. The inhibitory potential of these compounds against CHIKV has been performed in Vero cells using plaque reduction assay. EC50 of the compounds was calculated for L-7, WIN, SB2, NAL, DHD, GSK, and FLU which was found to be 1.996, 0.403, 5.387,1.528, 7.394, 3.664, and 0.618 μM, respectively. CHIKV RNA load has been further quantified using quantitative real-time PCR (qRT-PCR), which further validated the anti-CHIKV effect of the compounds. Immunofluorescence (IFA) studies have been conducted to analyze the antiviral potency of the compounds. Furthermore, IFA studies were performed to study the effect of the compounds on the SG assembly in the infected cells, therefore, to analyze any effect on the CHIKV replication. Thus, a relationship between the CHIKV replication and the virus-induced SGs has been established. Chapter 6 concludes the overall work of the thesis. The biochemical, crystallization, biophysical, and antiviral approaches on the NTF2-like domain of the G3BP1 are discussed in detail. The proposed compounds have the potential to be efficient inhibitors of viral-protein interaction and are validated to have an antiviral role against CHIKV.