Targeting the host protein G3BP1 for the discovery of novel antiviral inhibitors against Chikungunya virus.

The molecular interactions between Chikungunya virus (CHIKV) non-structural protein 3 (nsP3) and the host GTPase Activating SH3 Domain Binding Protein 1 (G3BP1) are critical for CHIKV replication. The C-terminus hypervariable domain (HVD) of nsP3 protein binds to the nuclear transport factor 2 (NTF2)-like domain of G3BP1 through two tandem FGDF motifs, aiding in the disruption of stress granule (SG) formation. Given G3BP1's role in the antiviral response, it presents an attractive target for antiviral drug development. In this study, seven potential small molecules targeting the FGDF motif binding pocket of G3BP1 were identified using a structure-based virtual screening approach. The binding modes of these molecules were further investigated through molecular docking and simulations. Surface Plasmon Resonance (SPR) and Isothermal Titration Calorimetry (ITC) experiments confirmed their binding to purified G3BP1 with micromolar (μM) affinity. The antiviral efficacy of these molecules was assessed using in vitro cell culture-based assays, revealing that L-7, WIN, SB2, NAL, DHD, GSK, and FLU effectively inhibited CHIKV replication with EC50 values of 1.99, 0.40, 5.38, 1.52, 7.39, 3.66, and 0.61 μM, respectively. Additionally, CHIKV-infected cells treated with these compounds exhibited fewer virus-induced SGs compared to untreated controls without affecting SG formation under oxidative stress conditions. These findings indicate that identified inhibitors successfully block G3BP1-nsP3 interactions and suppress CHIKV replication. This is one of the first reports of small antiviral molecules targeting G3BP1, a host protein essential for stress granule formation in the antiviral cellular response and CHIKV replication.